Terpene synthases from Santalum

ABSTRACT

An isolated nucleic acid molecule that encodes a terpene synthase and is selected from among: a) a nucleic acid molecule comprising the sequence of nucleotides set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5; b) a nucleic acid molecule that is a fragment of (a); c) a nucleic acid molecule comprising a sequence of nucleotides that is complementary to (a) or (b); and d) a nucleic acid molecule that encodes a terpene synthase having at least or at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to any one of (a)-(c); wherein the nucleic acid molecule encodes a terpene synthase.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/261,101, now allowed, filed Apr. 18, 2012, which is the NationalStage of International Application No. PCT/AU2010/000802, filed Jun. 25,2010, which claims benefit of priority to AU2009903016, filed Jun. 29,2009. The subject matter of the above-referenced applications areincorporated by reference herein.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ON COMPACT DISCS

An electronic version on compact disc (CD-R) of the Sequence Listing isfiled herewith in duplicate (labeled Copy #1 and Copy #2), the contentsof which are incorporated by reference in their entirety. Thecomputer-readable file on each of the aforementioned compact discs,created on Sep. 24, 2013, is identical, 32.4 kilobytes in size, andtitled 206BSEQ.US1.txt.

FIELD OF THE INVENTION

The present invention relates to a novel terpene synthase. The inventionfurther relates to nucleic acids encoding a terpene synthase, to methodsfor preparing variant terpene synthases, and to host-organismsexpressing the polypeptides of the invention. The present inventionfurther comprises methods for making a terpene synthase and methods formaking terpenoids such as terpenes.

BACKGROUND

The following discussion of the background art is intended to facilitatean understanding of the present invention only. The discussion is not anacknowledgement or admission that any of the material referred to is orwas part of the common general knowledge as at the priority date of theapplication.

Sandalwood, Santalum album (Santalaceae) is a small hemi-parasitictropical tree of great economic value found growing in southern India,Sri Lanka, eastern Indonesia and northern Australia. The timber ishighly sought after for its fine grain, high density and excellentcarving properties. Sandalwood timber contains resins and essentialoils, particularly the santalols, santalenes and dozens of other minorsesquiterpenoids. These chemicals provide the unique sandalwoodfragrance. The fragrant wood is usually ground and steam distilled, withthe essential oil serving as a fixative for many high-end perfumes.

Centuries of over-exploitation has led to the demise of sandalwood innatural stands. Large plantations are being established throughoutnorthern Australia to satisfy demand and conserve remaining reserves.Santalum album heartwood contains up to 6% dry wt. sesquiterpene oils,predominantly α- and β-santalol, α-trans-bergamotol and epi-β-santalol,along with the sesquiterpene olefins α- and β-santalene, α-bergamoteneand epi-β-santalene, β-bisabolene, α-, β- and γ-curcumene. The amount ofheartwood oil produced in a tree varies considerably, even undernear-identical growing conditions. Causes of this yield variation arenot well understood, but it is likely to be the result of both geneticand environmental factors.

Little is known about the biosynthesis of sesquiterpenoids such assesquiterpenes in S. album or how essential oil production is regulated.

The present invention addresses a need in the art for methods ofproducing terpenes similar to those produced by sandalwood.

SUMMARY

In one embodiment, the present invention provides an isolated nucleicacid molecule that encodes a terpene synthase and is selected fromamong:

(a) a nucleic acid molecule comprising the sequence of nucleotides setforth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5;

(b) a nucleic acid molecule that is a fragment of (a);

(c) a nucleic acid molecule comprising a sequence of nucleotides that iscomplementary to (a) or (b); and

(d) a nucleic acid molecule that encodes a terpene synthase having atleast or at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% identity to any one of (a)-(c);

wherein the nucleic acid molecule encodes a terpene synthase.

Other embodiments include: a polypeptide encoded by a nucleic acid ofthe invention; a host cell comprising a nucleic acid of the invention; anon-human organism modified to harbor a nucleic acid of the invention;and methods of producing a polypeptide comprising culturing host cellsof the invention.

In one embodiment, the invention provides a method of making at leastone terpene synthase comprising culturing a host cell modified tocontain at least one nucleic acid sequence under conditions conducive tothe production of said at least one terpene synthase.

The invention further provides an isolated terpene synthase, wherein theterpene synthase is a Santalum terpene synthase; and the terpenesynthase catalyzes the production of a santalene.

In another embodiment, the invention provides an isolated terpenesynthase, comprising:

(a) the sequence of amino acids set forth in SEQ ID NO: 2, SEQ ID NO: 4or SEQ ID NO: 6;

(b) the sequence of amino acids encoded by the nucleic acid molecule ofany of claims 1 to 5;

(c) a sequence of amino acids that is at least or at least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or more identical to the sequence ofamino acids set forth in SEQ ID NO:2; or

(d) a fragment of (a), (b) or (c);

wherein the terpene synthase catalyzes the production of a terpene.

The invention further provides a terpene synthase, wherein the terpenesynthase catalyzes the production of α-santalene, α-trans-bergamotene,epi-β-santalene and β-santalene concurrently.

The invention also provides methods for detecting the presence of aterpene synthase polypeptide or nucleic acid in a sample.

In a further embodiment, the invention provides a method of producing aterpene synthase, the method comprising the steps of:

(a) selecting a host organism and/or cell which does not express anucleic acid molecule having a sequence set forth in SEQ ID NO: 1, SEQID NO: 3 or SEQ ID NO: 5;

(b) transforming the organism with a nucleic acid molecule having asequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5; and

(c) culturing the organism under conditions conducive to the productionof the terpene synthase encoded by said nucleic acid.

In an alternative embodiment, the invention provides a method ofproducing a terpene synthase, the method comprising the steps of:

(a) selecting a host organism and/or cell which does express a nucleicacid molecule having a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3or SEQ ID NO: 5;

(b) transforming the organism with a nucleic acid molecule having asequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 inhigher quantity; and

(c) culturing the organism under conditions conducive to the productionof the terpene synthase encoded by said nucleic acid.

The invention further provides a method of making a terpene, comprising:

(a) contacting an acyclic pyrophosphate terpene precursor with theterpene synthase of the invention, and,

(b) optionally, isolating the terpene produced in step (a).

Preferably the method is performed on a terpene synthase that isheterologously expressed in a cell; wherein the acyclic pyrophosphateterpene precursor is expressed in the same cell as the terpene synthase;and wherein the step of contacting the acyclic pyrophosphate terpeneprecursor occurs in the cell. More preferably, the at least one terpeneis selected from among (+)-epi-β-santalene, (+)-β-santalene,(+)-β-santalene, (+)-α-santalene, (−)-α-santalene, cis-α-bergamotene,trans-α-bergamotene, trans-β-bergamotene and cis-β-bergamotene.

The terpenes produced by the terpene synthase of the present inventionmay be further processed to an alcohol, preferably α-santalol,β-santalol, α-trans-bergamotol and/or epi-β-santalol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Chromatogram of natural sandalwood oil from S. album. The 4 mainpeaks are α-santalene, E-α-bergamotene, epi-β-santalene and β-santalene.

FIGS. 2A and 2B: GC trace of SaSSy product profile after incubation withFPP, with mass spectrum data for α-santalene, α-trans-bergamotene,epi-β-santalene and β-santalene from the SaSSy product profile afterincubation with FPP as detected by GC-MS.

FIGS. 3A and 3B: Superposed GC traces of SaSSy, SauSSy and SspiSSyproduct profiles after incubation with FPP mass spectrum data forα-santalene, α-trans-bergamotene, epi-β-santalene, β-santalene,cis-β-farnesene and trans-β-farnesene from the SaSSy product profileafter incubation with FPP as detected by GC-MS.

FIG. 4: Nucleic acid sequence of the SaSSy terpene synthase of thepresent invention.

FIG. 5: Amino acid sequence of the SaSSy terpene synthase of the presentinvention.

FIGS. 6A and 6B: Clustal alignment of terpene synthases:FB299123-125—terpene synthase from Vetiveria zizanoides (WO2006/134523), AF484125—5-epi-aristolochene synthase from Nicotianaattenuata, AB438045—linalool synthase from Backhousia citriodora,Santalene—SaSSy santalene synthase of the present invention fromSantalum album.

FIG. 7: Phylogenetic tree of proteins aligned in FIG. 6.

FIG. 8: UPGMA alignment tree of 28 different terpene synthase genes.Reference codes as follows: 1 SaSSy [Santalum album]; 2 SauSSy [Santalumaustrocaledonicum]; 3 SspiSSy [Santalum spicatum]; 4 ACF24767.1monoterpene synthase [Santalum album]; 5 SauMonoTPS1 [Santalumaustrocaledonicum]; 6 SspiMonoTPS1 [Santalum spicatum]; 7 ACF24768.1SaSesquiTPS1 [Santalum album]; 8 SausSesquiTPS1 [Santalumaustrocaledonicum]; 9 SspiSesquiTPS1 [Santalum spicatum]; 10 AAS79351.1(−)-α-terpineol synthase [Vitis vinifera]; 11 BAG82825.1 linaloolsynthase [Backhousia citriodora]; 12 AAV63788.1 alpha-zingiberenesynthase [Ocimum basilicum]; 13 AAR99061.1 (−)-germacrene D synthase[Populus trichocarpa×Populus deltoides]; 14 CAA06614.15-epi-aristolochene synthase [Capsicum annuum var. annuum]; 15CAA77191.1 (+)-delta-cadinene synthase [Gossypium arboreum]; 16AAO73863.1 (+)-3-carene synthase [Picea abies]; 17 AAC05727.1 d-selinenesynthase [Abies grandis]; 18 AAF61453.1 beta-phellandrene synthase[Abies grandis]; 19 AAC05728.1 gamma-humulene synthase [Abies grandis];20 AAS47691.1 LAS [Picea abies]; 21 AAC39443.1 ent-kaurene synthase[Arabidopsis thaliana]; 22 ADB55710.1 (−)-ent-kaurene synthase [Piceasitchensis]; 23 AAM53944.1AF514287_(—)1 (+)-limonene synthase 1 [Citruslimon]; 24 AAA86337.1 vetispiradiene synthase [Hyoscyamus muticus]; 25AAF61439.1 amorpha-4,11-diene synthase [Artemisia annua]; 26 BAF02832.1monoterpene synthase [Eucalyptus globulus]; 27 Cineole synthase [Salviafruticosa]; 28 Sabinene synthase [Salvia pomifera].

FIG. 9: Neighbor joining alignment tree of 28 different terpene synthasegenes. Reference codes as for FIG. 8.

FIGS. 10 a-10 g: Alignment of nucleic acid sequences of terpenesynthases from S. austrocaledonicum, S. spicatum and S. album.

FIGS. 11 a-11 c: Alignment of amino acid sequences of terpene synthaseproteins from S. austrocaledonicum, S. spicatum and S. album.

FIGS. 12 a-12 d: Alignment of amino acid sequences of terpene synthaseproteins from S. austrocaledonicum, S. spicatum and S. album, comparedto the amino acid sequences of DQ785793.1—cineole synthase from Salviafruticosa and DQ785794.1—sabinene synthase from Salvia pomifera.

DETAILED DESCRIPTION

In accordance with the present invention, a novel terpene synthase genefrom S. album has been discovered, SaSSy. Orthologous genes from twoother phylogenetically divergent species (SspiSSy from S. spicatum andSauSSy from S. austrocaledonicum) were also found. The novel genes arecharacterised by the DNA sequences shown in SEQ ID NO: 1, SEQ ID NO: 3or SEQ ID NO: 5.

The novel gene disclosed in SEQ ID NO:1 is hereinafter generallyreferred to as SaSSy, the novel gene disclosed in SEQ ID NO:3 ishereinafter generally referred to as SauSSy, and the novel genedisclosed in SEQ ID NO:5 is hereinafter generally referred to asSspiSSy. The DNA and protein sequences of SaSSy, SauSSy and SspiSSy arevery highly conserved, with 94-98% identity over the amino acids of theORFs. Key domains of the gene are very highly conserved (see FIGS. 4 and5).

The novel terpene synthase enzyme catalyses the production from FPP ofterpenoids, preferably terpenes, more preferably sesquiterpenes chosenfrom the following: α-santalene, α-trans-bergamotene, epi-β-santaleneand β-santalene.

More specifically, SaSSy, SauSSy and SspiSSy are sesquiterpene synthasesand most specifically, santalene synthases. As used herein, a “terpenesynthase” is an enzyme that catalyses the production of one or moreterpenoids, or more preferably one or more terpenes from a substrate; a“sesquiterpene synthase” is an enzyme that catalyses the synthesis of asesquiterpenoid, or more preferably a sesquiterpene and a “santalenesynthase” is an enzyme that catalyses the synthesis of a santalene. Theformation of terpenoids and/or terpenes from a substrate can be assessedusing any method known in the art, including but not limited to, theenzyme assays and mass spectrometry described in Examples 6 and 7,below.

Terpenoids are defined herein as compounds derived from prenyldiphosphate substrates by activity of a terpene synthase and possiblesubsequent modification by other enzymes. Included with the termterpenoid are non-oxygenated terpene olefins (terpenes), oxygenatedterpenes, as well as other derivatives such as terpenols.

As used herein, a terpene is an unsaturated hydrocarbon based on theisoprene unit (C₅H₈), and having a general formula C₁₀H₁₆. Terpenes canbe acyclic, monocyclic or polycyclic. Terpenes include, but are notlimited to, monoterpenes, which contain 10 carbon atoms; sesquiterpenes,which contain 15 carbon atoms; diterpenes, which contain 20 carbonatoms, and triterpenes, which contain 30 carbon atoms. Reference to aterpene includes stereoisomers of the terpene.

Reference to a santalene in the present invention includes α-santaleneand β-santalene, and any stereoisomer thereof, including, for example,(+)-epi-β-santalene, (+)-β-santalene, (−)-β-santalene, (+)-α-santalene,and (−)-α-santalene.

This isolation of these novel terpene synthase genes will allow for thesynthesis of sandalwood oil which is similar to the natural oil. Todate, it is possible to synthesize some sesquiterpenes usingsesquiterpene synthases from other sources, but the range of componentsesquiterpenes and end ratio of each component is not similar to that ofthe natural sandalwood oil.

Preferably, the terpene synthase gene of the present invention isisolated from a member of the genus Santalum. More preferably, it isisolated from S. album (Indian Sandalwood, White Sandalwood, Chandana),S. spicatum (Australian Sandalwood) or S. austrocaledonicum. However,the gene may alternatively be isolated from a plant selected from thefollowing: S. acuminatum (Desert Quandong, Sweet Quandong, NativePeach); S. ellipticum (Coast Sandalwood); S. fernandezianum; S.freycinetianum; S. haleakalae; S. lanceolatum (Northern Sandalwood); S.macgregorii; S. murrayanum (Bitter Quandong); S. obtusifolium; S.paniculatum; S. salicifolium (Willowleaf Sandalwood); or S. yasi.

Thus there is provided an isolated terpene synthase, wherein the terpenesynthase is a Santalum terpene synthase, and the terpene synthasecatalyzes the production of a santalene.

Preferably, such a terpene synthase comprises:

(a) a sequence of amino acids sleeted from among the sequences set forthin SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO: 6; or

(b) a sequence of amino acids that is at least or at least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or more identical to the sequence ofamino acids set forth in SEQ ID NO:2; or;

(c) a fragment of (a) or (b); wherein

the terpene synthase catalyzes the production of a santalene.

In another embodiment, the invention provides an isolated terpenesynthase, comprising:

(a) the sequence of amino acids set forth in SEQ ID NO: 2, SEQ ID NO: 4or SEQ ID NO: 6;

(b) the sequence of amino acids encoded by the nucleic acid molecule ofany of claims 1 to 5;

(c) a sequence of amino acids that is at least or at least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or more identical to the sequence ofamino acids set forth in SEQ ID NO:2; or

(d) a fragment of (a), (b) or (c), where the terpene synthase catalyzesthe production of a terpene.

Preferably, such a terpene synthase comprises amino acids selected fromamong amino acids corresponding to positions 32-42, 221-425, 321-325,314-315 and 423-426 of SEQ ID NO:2.

More preferably, the terpene synthase catalyzes the production of aterpene selected from among monocyclic sequiterpenes, bicyclicsesquiterpenes and tricyclic sesquiterpenes, particularly wherein theterpene is synthesised from an acyclic pyrophosphate terpene precursorsuch as farnesyl-diphosphate (FPP).

The nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5encompasses a single ORF. The invention provides an isolated DNAnucleotide sequence corresponding to the terpene synthase gene SaSSynucleotide sequence depicted in SEQ ID NO:1, the SauSSy nucleotidesequence depicted in SEQ ID NO: 3 or the SspiSSy nucleotide sequencedepicted in SEQ ID NO: 5 or sequences substantially homologous to SEQ IDNO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, or fragments thereof. The inventionfurther provides a DNA sequence comprising the complement of SEQ ID NO:1, SEQ ID NO: 3 or SEQ ID NO: 5, or sequences substantially homologousto SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, or fragments thereof.

The invention therefore provides an isolated nucleic acid molecule thatencodes a terpene synthase and is selected from among:

(a) a nucleic acid molecule comprising the sequence of nucleotides setforth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5;

(b) a nucleic acid molecule that is a fragment of a);

(c) a nucleic acid molecule comprising a sequence of nucleotides that iscomplementary to (a)- or (b); and

(d) a nucleic acid molecule that encodes a terpene synthase having atleast or at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% identity to any one of (a)-(c);

wherein the nucleic acid molecule encodes a terpene synthase.

The isolated nucleic acid molecules that encode the terpene synthasepreferably have at least or at least about 61%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% identity to the terpene synthase encoded byany one of (a)-(d), wherein the differences in the sequence are aminoacid substitutions.

The nucleotide sequences of SaSSy, SauSSy and SspiSSy are very highlyconserved (see FIG. 4).

The nucleotide sequence of SaSSy, as set forth in SEQ ID NO:1, encodes apolypeptide that has a proline at amino acid position 143 (SEQ ID NO:2).However, the encoded polypeptide may alternatively have a serine atposition 143 (SEQ ID NO:7 and SEQ ID NO:8). The two variant sequencesare substantially homologous, particularly as the two residues are bothpolar, and subsequent tests have shown that when the two proteins areassayed they have substantially the same activity and produced eachcompound in substantially the same proportions. Generally, the twovariant proteins have identical activities and produced each compound inidentical proportions.

The DNA sequence may also correspond to a fragment of SEQ ID NO: 1, SEQID NO: 3 or SEQ ID NO: 5. Preferably, the fragment is selected from thefollowing locations of SEQ ID NO:1: position 961-975 (generallycorresponding to the DDxxD motif), position 94-126 (generallycorresponding to the R(R/P)X₈W motif). Without being bound to anyparticular theories, it is believed that the DDxxD motif is responsiblefor chelating the divalent metal ion (for example, magnesium) and if itis removed, the protein may be rendered completely non-functional. TheR(R/P)X₈W motif at the start of the protein is also believed to beunique to terpene synthases and is understood to be involved in thespecific ionisation of non-chiral FPP or GPP to chiral nerolidyl- andneryl diphosphate respectively.

Other regions which may be essential for the specific function of thethree santalene synthases of the present invention are amino acidpositions 314 and 315 (nucleotide positions 940 to 945), by inference ofthe work of Kampranis et al. (2007). Exchanging these residues withlarger or smaller residues of similar polarity is likely to change thesize of the active site, and hence the products produced upon catalysis.Amino acid residues 422 to 426 of the santalene synthases (nucleotides1264 to 1278) may also be responsible for the final product profile, asKampranis et al. demonstrated. The significance of the proline residueat the start of the α19 helix is to hold the substrate tightly, andremoval of this may cause a lack of functionality. Amino acid positions221-426 define a larger region encompassing many of the more importantregions that are preferably retained. Thus, residues in these locationsare preferably conserved for full function, or varied slightly foraltered function. Therefore, preferably the DNA sequence may alsocorrespond to a fragment of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5selected from the following locations of SEQ ID NO:1: position 940-945,661-1278 or 1264-1278.

Homologous nucleic acid molecules refer to a pre-determined number ofidentical or homologous nucleotides. Homology includes substitutionsthat do not change the encoded amino acid (i.e., “silent substitutions”)as well identical residues. Substantially homologous nucleic acidmolecules hybridize typically at moderate stringency or at highstringency all along the length of the nucleic acid or along at leastabout 70%, 80% or 90% of the full-length nucleic acid molecule ofinterest. Also contemplated are nucleic acid molecules that containdegenerate codons in place of codons in the hybridizing nucleic acidmolecule.

Whether any two nucleic acid molecules have nucleotide sequences thatare at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” can bedetermined using known computer algorithms such as the “FAST A” program,using for example, the default parameters as in Pearson et al. (Proc.Natl. Acad. Sci. USA 85:2444 (1988); other programs include the GCGprogram package (Devereux, J., et al., Nucleic Acids Research 12(I):387(1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J. Molec. Biol.215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., AcademicPress, San Diego (1994), and Carillo et al., SIAM J. Applied Math48:1073 (1988)). Other commercially or publicly available programsinclude DNAStar “MegAlign” program (Madison, Wis.) and the University ofWisconsin Genetics Computer Group (UWG) “Gap” program (Madison, Wis.)).Percent homology or identity of nucleic acid molecules can bedetermined, for example, by comparing sequence information using a GAPcomputer program (e.g., Needleman et al., J. Mol. Biol. 48: 443 (1970),as revised by Smith and Waterman (Adv. Appl. Math. 2:482 (1981)).Briefly, a GAP program defines similarity as the number of alignedsymbols (i.e., nucleotides) which are similar, divided by the totalnumber of symbols in the shorter of the two sequences. Defaultparameters for the GAP program can include: (1) a unary comparisonmatrix (containing a value of 1 for identities and 0 for non identities)and the weighted comparison matrix of Gribskov et al., Nucl. Acids Res.14:6745 (1986), as described by Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps.

Substantial homology or identity exists when polynucleotide sequence ofthe present invention, or fragment thereof, will specifically hybridiseto another SaSSy, SauSSy or SspiSSy polynucleotide (or a complementarystrand thereof) under selective hybridisation conditions. As usedherein, “specifically hybridises” refers to annealing, by complementarybase-pairing, of a nucleic acid molecule (e.g., an oligonucleotide) to atarget nucleic acid molecule. Those of skill in the art are familiarwith in vitro and in vivo parameters that affect specific hybridization,such as length and composition of the particular molecule. Parametersparticularly relevant to in vitro hybridization further includeannealing and washing temperature, buffer composition and saltconcentration. Exemplary washing conditions for removingnon-specifically bound nucleic acid molecules at high stringency are0.1×SSPE, 0.1% SDS, 65° C., and at medium stringency are 0.2×SSPE, 0.1%SDS, 50° C. Equivalent stringency conditions are known in the art. Theskilled person can readily adjust these parameters to achieve specifichybridization of a nucleic acid molecule to a target nucleic acidmolecule appropriate for a particular application, under conditions thatare low, medium or high stringency.

Typically, selective hybridisation will occur when there is at leastabout 55% identity over a stretch of at least about 14 nucleotides,preferably at least about 65%, more preferably at least about 75% andmost preferably at least about 90%. The length of homology comparison,as described, may be over longer stretches and in certain embodimentswill often be over a stretch of at least about nine nucleotides, usuallyat least about 20 nucleotides, more usually at least about 24nucleotides, typically at least about 28 nucleotides, more typically atleast about 32 nucleotides and preferably at least about 36 or morenucleotides.

Thus, the polynucleotide sequences of the invention preferably have atleast 75%, more preferably at least 85%, more preferably at least 90%homology to the sequences shown in the sequence listings herein. Morepreferably there is at least 95%, more preferably at least 98%,homology. Nucleotide homology comparisons may be conducted as describedbelow for polypeptides. A preferred sequence comparison program is theGCG Wisconsin Bestfit program.

In the context of the present invention, a homologous sequence is takento include a nucleotide sequence which is at least 60, 70, 80 or 90%identical, preferably at least 95 or 98% identical at the nucleic acidlevel over at least 20, 50, 100, 200, 300, 500, 1000, 1500 or 1710nucleotides with the corresponding nucleotide sequences set out in SEQID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5. In particular, homology shouldtypically be considered with respect to those regions of the nucleicacid sequence that encode contiguous amino acid sequences known to beessential for the function of the terpene synthase gene, rather thannon-essential neighboring sequences. For example, the nucleic acidsequence that codes for amino acid positions 32-42 (nucleotides 94-126)and/or amino acid positions 321-325 (nucleotides 961-975), and/oralternatively the nucleic acid sequence that codes for amino acidpositions 221-426 (nucleotides 661-1278), position 314-315 (nucleotides940-945) and/or position 422-426 (nucleotides 1264-1278).

SaSSy, SauSSy or SspiSSy polynucleotide sequence fragments of theinvention will preferably be at least 15 nucleotides in length, morepreferably at least 20, 30, 40, 50, 100 or 200 nucleotides in length.Generally, the shorter the length of the polynucleotide sequence, thegreater the homology required to obtain selective hybridisation.Consequently, where a polynucleotide sequence of the invention consistsof less than about 30 nucleotides, it is preferred that the percentageidentity is greater than 75%, preferably greater than 90% or 95%compared with the polynucleotide sequences set out in the sequencelistings herein. Conversely, where a polynucleotide sequence of theinvention consists of, for example, greater than 50 or 100 nucleotides,the percentage identity compared with the polynucleotide sequences setout in the sequence listings herein may be lower, for example greaterthan 50%, preferably greater than 60 or 75%.

Nucleic acid sequences according to the present invention which arehomologous to the sequences as represented by SEQ ID NO: 1, SEQ ID NO: 3or SEQ ID NO: 5 can be characterized and isolated according to any ofthe techniques known in the art, such as amplification by means ofsequence-specific primers, hybridization with sequence-specific probesunder more or less stringent conditions, serological screening methodsor via the LiPA typing system.

The genomic DNA sequence of SaSSy is provided in SEQ ID NO:9.

The RNA of the SaSSy, SauSSy and SspiSSy genes is also provided. The RNAsequence is preferably derived from the DNA sequences described aboveand provided in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5.

The invention also provides RNA fragments hybridisable with the genomicDNA of SaSSy, SauSSy or SspiSSy. The RNA or RNA fragment sequence mayalso be derived from the cDNA sequence of SaSSy, SauSSy or SspiSSy orfragments thereof.

Nucleic acid sequences and fragments, which include some deletions ormutations which would not substantially alter their ability to hybridizewith SaSSy, SauSSy or SspiSSy, are also provided by the presentinvention. Such variants are to be considered as forming obviousequivalents of the DNA, RNA or fragments referred to above.

Other preferred variant nucleic acid sequences of the present inventioninclude sequences which are redundant as a result of the degeneracy ofthe genetic code compared to any of the above-given nucleic acidsequences of the present invention. These variant nucleic acid sequenceswill thus encode the same amino acid sequences as the nucleic acidsequences they are derived from. Preferably, the DNA, RNA or cDNA ofthese variants are hybridisable to corresponding parts of the SaSSy,SauSSy or SspiSSy gene sequence.

Also included within the present invention are sequence variants of theDNA sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 orcorresponding RNA sequences or fragments thereof, containing eitherdeletions and/or insertions of one or more nucleotides, especiallyinsertions or deletions of 1 or more codons.

Also included are substitutions of some non-essential nucleotides byothers (including modified nucleotides and/or inosine).

Particularly preferred variant polynucleotides of the present inventionalso include sequences which hybridise under stringent conditions withany of the nucleic acid sequences of the present invention. Thus,sequences which show a high degree of homology (similarity) to any ofthe nucleic acid sequences of the invention as described above arepreferred. Particularly preferred are sequences which are at least 80%,85%, 90%, 95% or more homologous to said nucleic acid sequences of theinvention. Preferably, said sequences will have less than 20%, 15%, 10%,or 5% variation of the original nucleotides of said nucleic acidsequences.

Primer and probes are further provided, which can be made starting fromany DNA or RNA sequence or sequence fragment according to the invention.Preferably, such probes or primers are between about 5 to 50 nucleotideslong, more preferably from about 10 to 25 nucleotides. Preferably, theprobe or primer oligonucleotide contains at least or at least about 15,20, 25, 30, 35, 40, 45, 50, 60 or more contiguous nucleotides from aterpene synthase nucleic acid molecule. Probes and primers of thepresent invention may be used in PCR, sequencing reactions,hybridisation reactions and other applications known to the skilledperson. Preferably, the probes and/or primers will be generated fromregions of high G and C content, which are readily identified by theskilled addressee.

The present invention also relates to an oligonucleotide primercomprising part of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, saidprimer being able to act as a primer for specifically amplifying thenucleic acids of SaSSy, SauSSy or SspiSSy. Preferably, the primer is asingle stranded DNA oligonucleotide sequence capable of acting as apoint of initiation for synthesis of a primer extension product which iscomplementary to the nucleic acid strand to be copied. The specificlength and sequence of the primer used will depend on the complexity ofthe required DNA or RNA targets, as well as on the conditions of primeruse, such as temperature and ionic strength. The fact that amplificationprimers do not have to match exactly with corresponding templatesequence to warrant proper amplification is amply documented in theliterature (Kwok et al., 1990).

The amplification method used can be either polymerase chain reaction(PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al.,1988; Wu & Wallace, 1989; Barmy, 1991), nucleic acid sequence-basedamplification (NASBA; Guatelli et al., 1990; Compton, 1991),transcription-based amplification system (TAS; Kwoh et al., 1989),strand displacement amplification (SDA; Duck, 1990; Walker et al., 1992)or amplification by means of Qβ replicase (Lizardi et al., 1988; Lomeliet al., 1989) or any other suitable method to amplify nucleic acidmolecules using primer extension. During amplification, the amplifiedproducts can be conveniently labelled either using labelled primers orby incorporating labelled nucleotides. Labels may be isotopic (³²P, ³⁵S,etc.) or non-isotopic (biotin, digoxigenin, etc.). The amplificationreaction is repeated between 20 and 70 times, advantageously between 25and 45 times.

The present invention also relates to an oligonucleotide probecomprising part of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, with saidprobe being able to act as a hybridisation probe for the SaSSy, SauSSyor SspiSSy gene. Preferably, the probe is a single strandedsequence-specific oligonucleotide sequence which has a sequence that iscomplementary to the target sequence of the SaSSy, SauSSy or SspiSSygene to be detected.

Those skilled in the art will recognize that the stringency ofhybridisation will be affected by such conditions as salt concentration,temperature, or organic solvents, in addition to the base composition,length of the complementary strands and the number of nucleotide basemismatches between the hybridising nucleic acids. Stringent temperatureconditions will generally include temperatures in excess of 30° C.,typically in excess of 37° C., and preferably in excess of 45° C.Stringent salt conditions will ordinarily be less than 1000 mM,typically less than 500 mM, and preferably less than 200 mM. However,the combination of parameters is much more important than the measure ofany single parameter. An example of stringent hybridisation conditionsis 65° C. and 0.1×SSC (1×SSC=0.15 M NaCl, 0.015 M sodium citrate pH7.0).

Optionally, the probe of the invention is labelled and/or attached to asolid substrate. The solid substrate can refer to any substrate to whichan oligonucleotide probe can be coupled, provided that it retains itshybridization characteristics and provided that the background level ofhybridization remains low. Usually the solid substrate will be amicrotiter plate, a membrane (e.g., nylon or nitrocellulose) or amicrosphere (bead). Prior to application to the membrane or fixation itmay be convenient to modify the nucleic acid probe in order tofacilitate fixation or improve the hybridization efficiency. Suchmodifications may encompass homopolymer tailing, coupling with differentreactive groups such as aliphatic groups, NH₂ groups, SH groups,carboxylic groups, or coupling with biotin or haptens.

The probes of the invention may include also an isolated polynucleotideattached to a label or reporter molecule and may be used to isolateother polynucleotide sequences, having sequence similarity by standardmethods. For techniques for preparing and labelling probes see, e.g.,Sambrook and Russell (2001) or Ausubel et al. (2001).

Oligonucleotides according to the present invention and used as primersor probes may also contain or consist of nucleotide analogues such asphosphorothioates (Matsukura et al., 1987), alkylphosphonates (Miller etal., 1979) or peptide nucleic acids (Nielsen et al., 1991; Nielsen etal., 1993), or may contain intercalating agents (Asseline et al., 1984).The introduction of these modifications may be advantageous in order topositively influence characteristics such as hybridization kinetics,reversibility of the hybrid-formation, biological stability of theoligonucleotide molecules, etc.

Recombinant DNAs containing fragments of the DNA sequence of the SaSSy,SauSSy or SspiSSy genes are also provided by the present invention, andmay be used as, for example, probes. Preferably, the plasmid used togenerate the recombinant DNA is a plasmid amplifiable in prokaryotic oreukaryotic cells and carrying said fragments. For example, using clonedDNA containing a DNA fragment of the SaSSy gene as a molecularhybridization probe, either by marking with radionucleotides or withfluorescent reagents, the gene may be detected directly, for example, indifferent tissues of a sandalwood tree.

SaSSy, SauSSy or SspiSSy polynucleotide sequences (preferably in theform of probes) may also be immobilised to a solid phase support for thedetection of the SaSSy gene. In an alternate form of the invention,SaSSy, SauSSy or SspiSSy polynucleotide sequences together with otherpolynucleotide sequences (such as from other terpene synthase genes) maybe immobilized on a solid support in such a manner as to permitidentification of the presence of suitable terpene synthase genes and/orany of the other polynucleotide sequences bound onto the solid supportin a material such as a tree sample.

Techniques for producing immobilized libraries of DNA molecules havebeen described in the art. Generally, most prior art methods describethe synthesis of single-stranded nucleic acid molecule libraries, usingfor example masking techniques to build up various permutations ofsequences at the various discrete positions on the solid substrate. U.S.Pat. No. 5,837,832 describes an improved method for producing DNA arraysimmobilized to silicon substrates based on very large scale integrationtechnology. In particular, U.S. Pat. No. 5,837,832 describes a strategycalled “tiling” to synthesize specific sets of probes at spatiallydefined locations on a substrate that may be used to produce theimmobilized DNA libraries of the present invention. U.S. Pat. No.5,837,832 also provides references for earlier techniques that may alsobe used. Thus, polynucleotide sequence probes may be synthesised in situon the surface of the substrate.

Alternatively, single-stranded molecules may be synthesised off thesolid substrate and each pre-formed sequence applied to a discreteposition on the solid substrate. For example, polynucleotide sequencesmay be printed directly onto the substrate using robotic devicesequipped with either pins or piezoelectric devices.

The library sequences are typically immobilized onto or in discreteregions of a solid substrate. The substrate may be porous to allowimmobilization within the substrate or substantially non-porous, inwhich case the library sequences are typically immobilized on thesurface of the substrate. The solid substrate may be made of anymaterial to which polypeptides can bind, either directly or indirectly.Examples of suitable solid substrates include flat glass, siliconwafers, mica, ceramics and organic polymers such as plastics, includingpolystyrene and polymethacrylate. It may also be possible to usesemi-permeable membranes such as nitrocellulose or nylon membranes,which are widely available. The semi-permeable membranes may be mountedon a more robust solid surface such as glass. The surfaces mayoptionally be coated with a layer of metal, such as gold, platinum orother transition metal. A particular example of a suitable solidsubstrate is the commercially available BiaCore™ chip (PharmaciaBiosensors).

Preferably, the solid substrate is generally a material having a rigidor semi-rigid surface. In preferred embodiments, at least one surface ofthe substrate will be substantially flat, although in some embodimentsit may be desirable to physically separate regions for differentpolymers with, for example, raised regions or etched trenches. It isalso preferred that the solid substrate is suitable for the high densityapplication of DNA sequences in discrete areas of typically from 50 to100 μm, giving a density of 10000 to 40000 dots/cm⁻².

The solid substrate is conveniently divided up into sections. This maybe achieved by techniques such as photoetching, or by the application ofhydrophobic inks, for example teflon-based inks (Cel-line, USA).

Attachment of the polynucleotide sequences to the substrate may be bycovalent or non-covalent means. The polynucleotide sequences may beattached to the substrate via a layer of molecules to which the librarysequences bind. For example, the polynucleotide sequences may belabelled with biotin and the substrate coated with avidin and/orstreptavidin. A convenient feature of using biotinylated polynucleotidesequences is that the efficiency of coupling to the solid substrate canbe determined easily. Since the polynucleotide sequences may bind onlypoorly to some solid substrates, it is often necessary to provide achemical interface between the solid substrate (such as in the case ofglass) and the nucleic acid sequences. Examples of suitable chemicalinterfaces include hexaethylene glycol. Another example is the use ofpolylysine coated glass, the polylysine then being chemically modifiedusing standard procedures to introduce an affinity ligand. Other methodsfor attaching molecules to the surfaces of solid substrate by the use ofcoupling agents are known in the art, see for example WO 98/49557.

Binding of complementary polynucleotide sequences to the immobilizednucleic acid library may be determined by a variety of means such aschanges in the optical characteristics of the bound polynucleotidesequence (i.e., by the use of ethidium bromide) or by the use oflabelled nucleic acids, such as polypeptides labelled with fluorophores.Other detection techniques that do not require the use of labels includeoptical techniques such as optoacoustics, reflectometry, ellipsometryand surface plasmon resonance (see WO 97/49989).

Thus, the present invention provides a solid substrate havingimmobilized thereon at least one polynucleotide of the presentinvention, preferably two or more different polynucleotide sequences ofthe present invention. In a preferred embodiment the solid substratefurther comprises polynucleotide sequences derived from genes other thanthe SaSSy, SauSSy or SspiSSy polynucleotide sequences.

Preferably, the transcription of the SaSSy, SauSSy or SspiSSy genes isup-regulated or promoted, for example in an artificial system such ashost cells or recombinant plants, which may result in enhanced oilproduction. Such up-regulation or enhancement may be achieved by amyriad of means, such as inserting additional or alternative regulationsequences upstream and/or downstream of the genes in question, orgenerating mutants of the existing regulatory sequences which arecapable of increasing transcription due to, for example, increasedbinding of regulatory elements.

The invention also covers polypeptides encoded by the above RNA and DNAnucleotide sequences and fragments thereof. The invention furtherprovides an isolated SaSSy, SauSSy or SspiSSy amino acid sequence asshown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 and fragmentsthereof. More desirably, the SaSSy, SauSSy or SspiSSy amino acidsequences are provided in substantially purified form. Further providedare polypeptide fragments having lower molecular weights and havingpeptide sequences or fragments in common with those shown in SEQ ID NO:2, SEQ ID NO: 4 or SEQ ID NO: 6.

As used herein, an “amino acid” is an organic compound containing anamino group and a carboxylic acid group. A polypeptide contains two ormore amino acids. For purposes herein, amino acids include the twentynaturally-occurring amino acids, non-natural amino acids and amino acidanalogs (i.e., amino acids wherein the α-carbon has a side chain).

In keeping with standard polypeptide nomenclature described in J. Biol.Chem., 243:3557-3559 (1968), and adopted 37 C.F.R. §§1.821-1.822,abbreviations for the amino acid residues are shown in the below Tableof Correspondence:

SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyr Tyrosine G Gly Glycine F PhePhenylalanine M Met Methionine A Ala Alanine S Ser Serine I IleIsoleucine L Leu Leucine T Thr Threonine V Val Valine P Pro Proline KLys Lysine H His Histidine Q Gln Glutamine E Glu Glutamic acid Z Glx Gluand/or Gln W Trp Tryptophan R Arg Arginine D Asp Aspartic acid N AsnAsparagine B Asx Asn and/or Asp C Cys Cysteine X Xaa Unknown or other

It should be noted that all amino acid residue sequences representedherein by formulae have a left to right orientation in the conventionaldirection of amino-terminus to carboxyl-terminus. In addition, thephrase “amino acid residue” is broadly defined to include the aminoacids listed in the Table of Correspondence and modified and unusualamino acids, such as those referred to in 37 C.F.R. §§1.821-1.822, andincorporated herein by reference. Furthermore, it should be noted that adash at the beginning or end of an amino acid residue sequence indicatesa peptide bond to a further sequence of one or more amino acid residues,to an amino-terminal group such as NH₂ or to a carboxyl-terminal groupsuch as COOH.

As used herein, “naturally occurring amino acids” refer to the 20L-amino acids that occur in polypeptides, and “non-natural amino acid”refers to an organic compound containing an amino group and a carboxylicacid group that is not one of the naturally-occurring amino acids listedin the Table of Correspondence. Non-naturally occurring amino acids thusinclude, for example, amino acids or analogs of amino acids other thanthe 20 naturally-occurring amino acids and include, but are not limitedto, the D-isostereomers of amino acids. Non-naturally occurring aminoacids can be incorporated into the terpene synthases and variantsthereof provided herein.

The term “isolated” is used to describe an amino acid sequence of thepresent invention that has been separated from components that accompanyit in its natural state. Further, an amino acid sequence of the presentinvention is “substantially purified” when at least about 60 to 75% of asample exhibits a single SaSSy amino acid sequence. A substantiallypurified SaSSy, SauSSy or SspiSSy amino acid sequence will typicallycomprise about 60 to 90% W/W of a sample, more usually about 95%, andpreferably will be over about 99% pure. Protein purity or homogeneitymay be indicated by a number of means well known in the art, such aspolyacrylamide gel electrophoresis of a protein sample, followed byvisualizing a single amino acid sequence band upon staining the gel. Forcertain purposes, higher resolution may be provided by using HPLC orother means well known in the art which are utilised for application.

The invention further contemplates fragments of the SaSSy, SauSSy orSspiSSy amino acid sequences. An amino acid sequence fragment inaccordance with this aspect of the invention is a stretch of amino acidresidues of at least about five to seven contiguous amino acids, oftenat least about seven to nine contiguous amino acids, typically at leastabout nine to 13 contiguous amino acids and, most preferably, at leastabout 20 to 30 or more contiguous amino acids.

In a highly preferred form of the invention, the fragments exhibitligand-binding, immunological activity and/or other biologicalactivities characteristic of SaSSy, SauSSy or SspiSSy amino acidsequences. More preferably, the fragments possess immunological epitopesconsistent with those present on native SaSSy, SauSSy and SspiSSy aminoacid sequences.

As used herein, “epitope” refers to an antigenic determinant of apolypeptide. An epitope could comprise three amino acids in a spatialconformation that is unique to the epitope. Generally, an epitopeconsists of at least five amino acids, and more usually consists of atleast 8-10 amino acids. Methods of determining the spatial conformationof such amino acids are known in the art.

Preferred SaSSy, SauSSy or SspiSSy amino acid sequences of the inventionwill have one or more biological properties (e.g., in vivo, in vitro orimmunological properties) of the native full-length amino acid sequence.Alternatively, fragments of the full-length SaSSy, SauSSy or SspiSSyamino acid sequences may have one or more biological properties of thegenes which the full length amino acid sequence encodes.

Antibodies to specific regions of the SaSSy, SauSSy or SspiSSy genes maybe determined by those skilled in the art, using, for example, thetechniques recited in: G. W. Turner, and R. Croteau, “Organization ofmonoterpene biosynthesis in Mentha. Immunocytochemical localizations ofgeranyl diphosphate synthase, limonene-6-hydroxylase, isopiperitenoldehydrogenase, and pulegone reductase,” Plant Physiol. 136:4215-4227(2004).

Amino acid sequences, including analogues, fragments and derivatives, ofthe SaSSy, SauSSy or SspiSSy genes can be prepared synthetically (e.g.,using the well known techniques of solid phase or solution phase peptidesynthesis). Preferably, solid phase synthetic techniques are employed.Alternatively, SaSSy, SauSSy or SspiSSy amino acid sequences of theinvention can be prepared using well known genetic engineeringtechniques, as described infra. In yet another embodiment, the aminoacid sequences can be purified (e.g., by immunoaffinity purification)from a biological material such as tree heartwood, sap, etc.

Amino acid sequences and derived peptide biomarkers to specific regionsof the SaSSy, SauSSy or SspiSSy genes may be determined by those skilledin the art, using, for example, the techniques recited in: Zulak, K. G.,Lippert, D. N., Kuzyk, M., Domanski, D., Chou, T., Borchers, C. H. andJ. Bohlmann, “Targeted proteomics using selected reaction monitoring(SRM) reveals the induction of specific terpene synthases in amulti-level study of methyl jasmonate treated Norway spruce (Piceaabies),” The Plant Journal 60:1015-1030 (2009).

SaSSy, SauSSy or SspiSSy amino acid sequence analogues preferablyinclude those having an amino acid sequence wherein one or more of theamino acids is substituted with another amino acid, which substitutionsdo not substantially alter the biological activity of the molecule.

Variants of the SaSSy, SauSSy or SspiSSy terpene synthases of theinvention may be used to attain desired enhanced or reduced enzymaticactivity, modified regiochemistry or stereochemistry, or alteredsubstrate utilization or product distribution. A variant or site directmutant may be made by any methods known in the art. Variants andderivatives of native polypeptides can be obtained by isolatingnaturally-occurring variants, or the nucleotide sequence of variants, ofother or same plant lines or species, or by artificially programmingmutations of nucleotide sequences coding for native sandalwoodpolypeptides.

In the context of the invention, a homologous sequence is taken toinclude a SaSSy, SauSSy or SspiSSy amino acid sequence which is at least60, 70, 80 or 90% homologous, preferably at least 95 or 98% homologousat the amino acid level over at least 20, 50, 100, 200 or 570 aminoacids, with the amino acid sequence set out in SEQ ID NO: 2, SEQ ID NO:4 or SEQ ID NO: 6. In particular, homology should typically beconsidered with respect to those regions of the sequence that encodecontiguous amino acid sequences known to be essential for the functionof the terpene synthase gene, rather than non-essential neighbouringsequences. For example, amino acid positions 32-42 and/or amino acidpositions 321-325, or alternatively amino acid positions 221-425,positions 314-315 and/or positions 422-426.

Although homology can be considered in terms of similarity (i.e., aminoacid residues having similar chemical properties/functions), in thecontext of the present invention it is preferred to express homology interms of sequence identity. The terms “substantial homology” or“substantial identity,” when referring to SaSSy amino acid sequences,indicate that the amino acid sequence in question exhibits at leastabout 70% identity with an entire naturally-occurring SaSSy, SauSSy orSspiSSy amino acid sequence or portion thereof, usually at least about80% identity and preferably at least about 90 or 95% identity.

As used herein, “sequence identity” refers to the number of identicalamino acids (or nucleotide bases) in a comparison between a test and areference polypeptide or polynucleotide. “Homologous polypeptides” referto a pre-determined number of identical or homologous amino acidresidues. Homology includes conservative amino acid substitutions aswell as identical residues. Sequence identity can be determined bystandard alignment algorithm programs used with default gap penaltiesestablished by each supplier.

For determination of homology of proteins, conservative amino acids canbe aligned as well as identical amino acids; in this case, percentage ofidentity and percentage homology varies. Whether any two polypeptideshave amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% “identical” can be determined using known computeralgorithms such as the “FAST A” program, using for example, the defaultparameters as in Pearson et al. (Proc. Natl. Acad. Sci. USA 85:2444(1988); other programs include the GCG program package (Devereux, J., etal., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA(Atschul, S. F., et al., J. Molec. Biol. 215:403 (1990); Guide to HugeComputers, Martin J. Bishop, ed., Academic Press, San Diego (1994), andCarillo et al., SIAM J. Applied Math 48:1073 (1988)). For example, theBLAST function of the National Center for Biotechnology Informationdatabase can be used to determine identity. Other commercially orpublicly available programs include DNAStar “MegAlign” program (Madison,Wis.) and the University of Wisconsin Genetics Computer Group (UWG)“Gap” program (Madison, Wis.)).

Percent homology or identity of proteins and/or nucleic acid moleculescan be determined, for example, by comparing sequence information usinga GAP computer program (e.g., Needleman et al., J. Mol. Biol. 48:443(1970), as revised by Smith and Waterman (Adv. Appl. Math. 2:482(1981)). Briefly, a GAP program defines similarity as the number ofaligned symbols (i.e., nucleotides or amino acids) which are similar,divided by the total number of symbols in the shorter of the twosequences. Default parameters for the GAP program can include: (1) aunary comparison matrix (containing a value of 1 for identities and 0for non identities) and the weighted comparison matrix of Gribskov etal., Nucl. Acids Res. 14:6745 (1986), as described by Schwartz andDayhoff, eds., Atlas of Protein Sequence and Structure, NationalBiomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0for each gap and an additional 0.10 penalty for each symbol in each gap;and (3) no penalty for end gaps.

Therefore, as used herein, the term “identity” represents a comparisonbetween a test and a reference polypeptide or polynucleotide. In onenon-limiting example, “at least 90% identical to” refers to percentidentities from 90 to 100% relative to the reference polypeptides.Identity at a level of 90% or more is indicative of the fact that,assuming for exemplification purposes a test and referencepolynucleotide length of 100 amino acids are compared, no more than 10%(i.e., 10 out of 100) of amino acids in the test polypeptide differsfrom that of the reference polypeptides. Such differences can berepresented as point mutations randomly distributed over the entirelength of an amino acid sequence or they can be clustered in one or morelocations of varying length up to the maximum allowable, e.g., 10/100amino acid difference (approximately 90% identity). Differences aredefined as amino acid substitutions, insertions or deletions. At thelevel of homologies or identities above about 85-90%, the result shouldbe independent of the program and gap parameters set; such high levelsof identity can be assessed readily, often without relying on software.

In a highly preferred form of the invention, a SaSSy, SauSSy or SspiSSyamino acid sequence analogue will have 80% or greater amino acidsequence identity to the SaSSy, SauSSy or SspiSSy amino acid sequenceset out in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. Examples of suchamino acid sequence analogues within the scope of the invention includethe amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6wherein: (a) one or more aspartic acid residues is substituted withglutamic acid; (b) one or more isoleucine residues is substituted withleucine; (c) one or more glycine or valine residues is substituted withalanine; (d) one or more arginine residues is substituted withhistidine; (e) one or more tyrosine or phenylalanine residues issubstituted with tryptophan; or (f) one or more proline residues aresubstituted with serine residues.

Naturally-occurring peptide variants are also encompassed by theinvention. Examples of such variants are proteins that result fromalternate mRNA splicing events or from proteolytic cleavage of thepolypeptides described herein. Variations attributable to proteolysisinclude, for example, differences in the N- or C-termini upon expressionin different types of host cells, due to proteolytic removal of one ormore terminal amino acids from the polypeptides encoded by the sequencesof the invention.

SaSSy, SauSSy or SspiSSy amino acid sequence derivatives are alsoprovided by the invention and include amino acid sequences, analogues orfragments thereof which are substantially homologous in primarystructure but which include chemical and/or biochemical modifications orunusual amino acids. Such modifications include, for example,acetylation, carboxylation, phosphorylation, glycosylation,hydroxylation, sulfation, ubiquitination, labelling (e.g., withradionucleotides), and various enzymatic modifications, as will bereadily appreciated by those well skilled in the art.

The terpene synthases of the present invention may also be provided inthe form of a “chimeric protein” or “fusion protein.” Such proteins arepolypeptides operatively-linked to a different polypeptide. A chimericor fusion protein provided herein can include one or more terpenesynthase polypeptides, or a portion thereof, and one or more otherpolypeptides for any one or more of a transcriptional/translationalcontrol signals, signal sequences, a tag for localization, a tag forpurification, part of a domain of an immunoglobulin G, and/or atargeting agent. These chimeric or fusion proteins include thoseproduced by recombinant means as fusion proteins, those produced bychemical means, such as by chemical coupling, through, for example,coupling to sulfhydryl groups, and those produced by any other methodwhereby at least one polypeptide (i.e., terpene synthase), or a portionthereof, is linked, directly or indirectly via linker(s) to anotherpolypeptide.

Where the SaSSy, SauSSy or SspiSSy amino acid sequences are to beprovided in a labelled form, a variety of methods for labelling aminoacid sequences are well known in the art and include radioactiveisotopes such as ³H or ¹⁴C, ligands which bind to labelled anti-ligands(e.g., antibodies), fluorophores, chemiluminescent agents, enzymes andanti-ligands which can serve as specific binding pair members for alabelled ligand. The choice of label depends on the sensitivityrequired, stability requirements, and available instrumentation. Methodsof labelling amino acid sequences are well known in the art [See, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989);and Ausubel, F., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J.G., Smith, J. A., Struhl, K., Current protocols in molecular biology,Greene Publishing Associates/Wiley Intersciences, New York (2001)].

The SaSSy, SauSSy and SspiSSy amino acid sequences of the invention, ifsoluble, may be coupled to a solid-phase support, e.g., nitrocellulose,nylon, column packing materials (e.g., Sepharose beads), magnetic beads,glass wool, plastic, metal, polymer gels, cells, or other substrates.Such supports may take the form, for example, of beads, wells,dipsticks, or membranes.

The invention also provides for fusion polypeptides, comprising SaSSy,SauSSy or SspiSSy amino acid sequences and fragments. Thus SaSSy, SauSSyor SspiSSy amino acid sequences may be fusions between two or more aminoacid sequences of the present invention or between an amino acidsequence from SaSSy, SauSSy or SspiSSy and a related protein. Likewise,heterologous fusions may be constructed which would exhibit acombination of properties or activities of the derivative proteins. Forexample, ligand-binding or other domains may be “swapped” betweendifferent fusion polypeptides or fragments. Such homologous orheterologous fusion polypeptides may display, for example, alteredstrength or specificity of binding. Fusion partners includeimmunoglobulins, bacterial beta-galactosidase, trpE, protein A,beta-lactamase, alpha amylase, alcohol dehydrogenase and yeast alphamating factor.

Modified SaSSy, SauSSy or SspiSSy amino acid sequences may besynthesised using conventional techniques, or may be encoded by amodified polynucleotide sequence and produced using recombinant nucleicacid methods. The modified polynucleotide sequence may also be preparedby conventional techniques. Fusion proteins will typically be made byeither recombinant nucleic acid methods or may be chemicallysynthesised.

Variants of the terpene synthases of the invention may be used to attaindesired enhanced or reduced enzymatic activity, modified regiochemistryor stereochemistry, or altered substrate utilization or productdistribution. Furthermore, variants may be prepared to have at least onemodified property, for example an increased affinity for the substrate,an improved specificity for the production of one or more desiredcompounds, a different product distribution, a different enzymaticactivity, an increase of the velocity of the enzyme reaction, a higheractivity or stability in a specific environment (pH, temperature,solvent, etc.), or an improved expression level in a desired expressionsystem. A variant or site direct mutant may be made by any method knownin the art.

As stated above, the invention provides recombinant and non-recombinant,isolated and purified polypeptides. Variants and derivatives of nativepolypeptides can be obtained by isolating naturally-occurring variants,or the nucleotide sequence of variants, of other or same plant lines orspecies, or by artificially programming mutations of nucleotidesequences coding for native terpene synthases. Alterations of the nativeamino acid sequence can be accomplished by any of a number ofconventional methods.

Polypeptide variants resulting from a fusion of additional peptidesequences at the amino and carboxyl terminal ends of the polypeptides ofthe invention can be used to enhance expression of the polypeptides, aidin the purification of the protein or improve the enzymatic activity ofthe polypeptide in a desired environment or expression system. Suchadditional peptide sequences may be signal peptides, for example.Accordingly, the present invention encompasses variants of thepolypeptides of the invention, such as those obtained by fusion withother oligo- or polypeptides and/or polypeptides which are linked tosignal peptides.

Therefore, in an embodiment, the present invention provides a method forpreparing a variant polypeptide having a desired terpene synthaseactivity, the method comprising the steps of:

(a) selecting any of the nucleic acids from the group consisting of SEQID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, fragments or variants thereof asdescribed above;

(b) modifying the selected nucleic acid to obtain at least one mutantnucleic acid;

(c) transforming host cells with the mutant nucleic acid sequence toexpress a polypeptide encoded by the mutant nucleic acid sequence;

(d) screening the polypeptide for a functional polypeptide having atleast one modified property; and,

(e) optionally, if the polypeptide has no desired variant terpenesynthase activity, repeat the process steps (a) to (d) until apolypeptide with a desired variant terpene synthase activity is obtained(i.e., DNA shuffling).

In step (b), a large number of mutant nucleic acid sequences may becreated, for example by random mutagenesis, site-specific mutagenesis,or DNA shuffling. The detailed procedures of gene shuffling are found inStemmer, W. P., “DNA shuffling by random fragmentation and reassembly:in vitro recombination for molecular evolution,” Proc. Natl. Acad. Sci.U.S.A. 91(22):10747-1075 (1994). In short, DNA shuffling refers to aprocess of random recombination of known sequences in vitro, involvingat least two nucleic acids selected for recombination. For examplemutations can be introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene whereinpredetermined codons can be altered by substitution, deletion orinsertion.

Accordingly, any of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5,fragments and variants thereof may be recombined with a differentsequence selected from any of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO:5, fragments and variants thereof, and/or with other terpene synthasesencoding nucleic acids, for example isolated from an organism other thanS. album, S. austrocaledonicum or S. spicatum. Thus, mutant nucleicacids may be obtained and separated, which may be used for transforminga host cell according to standard procedures.

In step (d), the polypeptide obtained in step (c) is screened for amodified property, for example a desired modified enzymatic activity.Examples of desired enzymatic activities for which an expressedpolypeptide may be screened include enhanced or reduced enzymaticactivity, as measured by K_(M) or V_(max) value, for example, modifiedregio-chemistry or stereochemistry, altered substrate utilization orproduct distribution. The screening of enzymatic activity can beperformed according to procedures familiar to the skilled person.Methods for determining kinetic data and analysis of terpene productsare given in, for example, Examples 13 and 14.

Step (e) provides for repetition of process steps (a)-(d), which may,preferably, be performed in parallel. Accordingly, by creating asignificant number of mutant nucleic acids, many host cells may betransformed with different mutant nucleic acids at the same time,allowing for the subsequent screening of an elevated number ofpolypeptides. The chances of obtaining a desired variant polypeptide maythus be increased at the discretion of the skilled person.

In an embodiment, the present invention provides a method for preparinga nucleic acid encoding a variant polypeptide having terpene synthaseactivity, the method comprising the steps (a)-(e) disclosed above andfurther comprising the step of:

(f) if a polypeptide having desired variant terpene synthase activitywas identified, acquiring the mutant nucleic acid obtained in step (c),which was used to transform host cells to express the variant terpenesynthase following steps (c) and (d).

Preferably, the terpene synthase polypeptides of the invention catalysethe production of mono-, bi- and/or tricyclic sesquiterpenes.Preferably, the terpene synthase produces or synthesises thesesquiterpenes from an acyclic pyrophosphate terpene precursorsubstrate. An acyclic pyrophosphate terpene precursor is any acyclicpryrophosphate compound that is a precursor to the production of atleast one terpene including, but not limited to, geranyl-pyrophosphate(GPP), farnesyl-diphosphate (FPP) and geranylgeranyl-pyrophosphate(GGPP). Generally, GPP is the precursor for the monoterpenes, FPP forthe sesquiterpenes, and GGPP for the diterpenes. Preferably, theprecursor is FPP.

As used herein, “catalyses the production of terpene(s)” or “catalysesthe production of santalene(s)” refers to the ability of a terpenesynthase to produce a terpene or a mixture thereof or specificallysantalene or a mixture thereof, respectively, from a substrate, such asan acyclic terpene precursor. The formation of terpenes, such assantalene, from a substrate by a terpene synthase can be assessed usingany method known in the art, including, but not limited to, the enzymeassays and mass spectrometry methods described in Examples 6 and 7,below.

Typically, a polypeptide that “catalyses the production of terpenes”produces a santalene or mixture thereof in an amount that is at least orat least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of the totalterpenes, by weight, from a particular substrate as measured using an invitro assay, such as those assays described in Examples 6, 7, 13 and 14,wherein the terpene synthase, such as a santalene synthase, is mixedwith a substrate, such as FPP, in the presence of Mg²⁺. The santalenesproduced by the synthase can include α-santalene and/or β-santalene,including (+)-epi-β-santalene, (+)-β-santalene, (−)-β-santalene,(+)-α-santalene, and (−)-α-santalene. For example, included among theterpene synthases provided herein are Santalum species terpene synthasesthat catalyze the production of α-santalene from FPP, wherein the amountof α-santalene produced from FPP at least is or is at least about 1%,2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90% or more of the total terpenes (by weight)produced from FPP. In other examples, the terpene synthase catalyses theproduction of β-santalene from FPP, wherein the amount of β-santaleneproduced from FPP is at least or is at least about 1%, 2%, 3%, 4%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90% or more of the total terpenes (by weight) produced fromFPP.

Preferably, the isolated polypeptides of the present invention arecapable of forming a bisabolyl cation from FPP and capable of furthercreating a bond between the C₃ and the C₇ carbon atom of FPP to producea bicyclic or tricyclic sesquiterpene comprising a C₃-C₇ bond.Similarly, the polypeptide of the present invention is capable offorming a bisabolyl cation from FPP and capable of further creating abond between the C₂ and the C₇ carbon atom of FPP to produce a bicyclicor tricyclic sesquiterpene comprising a C₂-C₇ bond.

The term “capable of synthesising” a compound, such as a specificsesquiterpene, and the terms “terpene synthase activity,” preferably“sesquiterpene synthase activity,” refers to polypeptides of the presentinvention, as well as nucleic acids encoding these polypeptides, whichare capable of synthesizing a terpene, preferably a sesquiterpene, morepreferably a santalene and most preferably the sesquiterpene and/orsantalene compounds mentioned herein from at least one startingcompound, which preferably is an acyclic pyrophosphate terpeneprecursor. Preferably, the acyclic terpene precursor is FPP which isgiven in the formula (I) below with standard numeration of the carbonskeleton of sesquiterpenes. OPP refers to pyrophosphate.

Preferably, the isolated terpene synthase polypeptides are capable ofsynthesising at least one sesquiterpene, more preferably at least onesesquiterpene having a santalene or bergamotene carbon skeleton. In apreferred embodiment, the polypeptide is capable of forming a bisabolylcation from FPP, and capable of further creating a bond between the C₃or C₂ and the C₇ carbon atom of FPP to produce one or several bi-cyclicand/or tricyclic sesquiterpenes.

The term “bond” refers to a single covalent bond.

The present invention relates to nucleic acids encoding polypeptides, aswell as to the polypeptides themselves, capable of synthesising at leastone bicyclic and/or tricyclic sesquiterpene comprising a C₃-C₇ bond.Preferably, the sesquiterpenes comprising a C₃-C₇ bond constitute atleast about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 80% or more of the total amount ofsesquiterpene products synthesised by the terpene synthase. Thequantitative sesquiterpene product distribution of a terpene synthase,for the purpose of the present invention, may be determined by employingthe procedure detailed in Example 7, or by determination of the % bycomposition using techniques well known to those skilled in the art.

Accordingly, the present invention relates to isolated polypeptidescapable of forming compounds having a C₃-C₇ bond of formulae (II) and/or(III) below,

in which R₁, R₂, R₃, R₄ are, independently of each other a linear orbranched alkyl or alkylene group from C₁ to C₂₀, and whereby R₁ and R₂and/or R₃ and R₄ may form a double bond instead of two individual singlebonds.

Preferably, R₁, R₂, R₃, and R₄ are, independently of each other, alinear or branched alkyl or alkylene group from C₁ to C₁₅, morepreferably from C₁ to C₁₀, most preferably, from C₁ to C₈.

In particular, the polypeptides of the present invention are capable offorming compounds of formulae (IV), (V) and/or (VI) below,

in which R₁, R₂, R₃, R₄ are defined as above.

Preferably, in formula (IV) and/or (VI), either R₁ or R₂ is a C₁-C₅alkyl and the other is a C₂-C₈ alkylene. In addition, R₃ in formula (VI)preferably is a C₁-C₅, more preferably a C₁-C₃ alkyl. Preferably, informula (V), R₃ and R₄ are defined as R₁ and R₂ in formula (IV) above.Most preferably, R₃ of compound VI is a methyl group and R₁ and R₂ arealternately a methyl and a C₆ linear alkyl group.

Preferably the sesquiterpene is a santalene, more preferably anα-santalene or β-santalene. The santalene may be a stereoisomer selectedfrom among (+)-epi-β-santalene, (+)-β-santalene, (−)-β-santalene,(+)-α-santalene, and (+)-α-santalene.

Preferably, the terpene synthase catalyzes the production ofα-santalene, and the amount of α-santalene produced is at least or atleast about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 80% or more of the total amount of terpeneproduced.

The present invention relates to nucleic acids encoding a polypeptide,and to the polypeptide itself, capable of forming at least onesesquiterpene having a C₂-C₇ bond. According to a preferred embodiment,sesquiterpenes comprising a C₂-C₇ bond constitute at least about 1%, 2%,3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 80% or more of the total amount of sesquiterpene productssynthesised by the terpene synthase.

The sesquiterpenes may be bergamotene, santalene and/or one of theisomers of these compounds, preferably stereoisomers. Preferably, theproduct is predominantly one stereoisomer, although it may furthercomprise a lesser amount of the other stereoisomer or enantiomer.

According to an embodiment, the present invention relates to isolatedpolypeptides capable of forming compounds having a C₂-C₇ bond accordingto the formulae (VII) and/or (VIII) below,

in which R₅ and R₆ are defined as R₁ and R₂ above. Preferably, R₅ is amethyl and R₆ is a C₂-C₁₀ alkenyl, or vice versa.

Preferably, at least one alkenyl possibly present in one of the residuesR₁, R₂, R₃, R₄, R₅ or R₆ mentioned above is 4-methyl-3-pentenyl, whileanother residue linked to the same carbon atom is methyl.

The polypeptides capable of synthesizing the compounds of formulae (II),(III), (IV), (V), (VI), (VII) and/or (VIII) above preferably are thepolypeptides having the amino acid sequences of SEQ ID NO: 2, SEQ ID NO:4 or SEQ ID NO: 6, or polypeptide variants thereof.

Preferably, the sesquiterpenes having a C₂-C₇ bond are bergamotene,including its stereoisomers, in particular, cis-α-bergamotene,trans-α-bergamotene, trans-β-bergamotene and cis-β-bergamotene, forexample.

Preferably, the terpene synthase catalyzes the production ofbergamotene, and the amount of bergamotene produced is at least or atleast about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 80% or more of the total amount of terpeneproduced.

In a further aspect, the invention provides isolated polypeptidescapable of synthesizing santalene and/or bergamotene. In a preferredembodiment, the invention provides isolated polypeptides capable ofsynthesizing one or more of the following: α-santalene,α-trans-bergamotene, epi-β-santalene, β-santalene andtrans-trans-farnesol. Preferably, the isolated polypeptides are capableof synthesising at least β-santalene.

Most preferably, the SaSSy, SauSSy and SspiSSy terpene synthases of thepresent invention are able to synthesise at least two or more terpenes,preferably a terpene selected from among (+)-epi-β-santalene,(−)-β-santalene, (+)-β-santalene, (+)-α-santalene, and (−)-α-santalene,E-α-bergamotene, γ-curcumene, β-bisabolene, β-curcumene, α-curcumene,trans-trans-farnesol, α-pinene, camphene, limonene and α-terpinolene,more preferably α-santalene, α-trans-bergamotene, epi-β-santalene andβ-santalene. Most preferably, the terpene synthase can synthesise two ormore of the above compounds concurrently.

The enzymes may preferably be able to synthesise at least the fourcompounds listed above in the same reaction mixture under the samereaction conditions. Thus, it is preferable that the enzymes are able toconvert FFP to at least the four listed terpenes under a single set ofconditions, without need for further input of different startingcompounds, different additional factors or alteration of reactionconditions to obtain one or more of the terpenes.

In a further aspect, the invention provides a vector comprising anucleic acid of the invention.

A “vector,” as used herein, includes prokaryotic vectors, viral vectors,or eukaryotic vectors and/or any recombinant vectors including, but notlimited to, bacteriophages and plasmids. The vector may further be anexpression vector. An expression vector includes vectors capable ofexpressing DNA that is operatively linked with regulatory sequences,such as promoter regions, that are capable of effecting expression ofsuch DNA fragments. Such additional segments can include promoter andterminator sequences, and optionally can include one or more origins ofreplication, one or more selectable markers, an enhancer, apolyadenylation signal, and the like. Expression vectors are generallyderived from plasmid or viral DNA, or can contain elements of both.Thus, an expression vector refers to a recombinant DNA or RNA construct,such as a plasmid, a phage, recombinant virus or other vector that, uponintroduction into an appropriate host cell, results in expression of thecloned DNA. Appropriate expression vectors are well known to those ofskill in the art and include those that are replicable in eukaryoticcells and/or prokaryotic cells and those that remain episomal or thosewhich integrate into the host cell genome. Viral vectors are engineeredviruses that are operatively linked to exogenous genes to transfer (asvehicles or shuttles) the exogenous genes into cells.

The skilled person is capable of selecting a suitable vector accordingto the expression system. In one embodiment, the expression vectorsinclude a cDNA sequence encoding the polypeptide operably linked toregulatory sequences such as transcriptional promoters, operators, orenhancers, mRNA ribosomal binding sites, and appropriate sequences whichcontrol transcription and translation initiation and termination forexample.

Nucleotide sequences are “operably linked” when the regulatory sequencefunctionally relates to the cDNA sequence of the invention. Operably oroperatively linking DNA segments means that the segments are arranged sothat they function in concert for their intended purposes, e.g.,transcription initiates in the promoter and proceeds through the codingsegment to the terminator.

The vectors of the present invention may be used in the methods forpreparing a genetically modified host organisms and/or cells, in hostorganisms and/or cells harbouring the nucleic acids of the invention andin the methods for producing or making terpene synthases, as is set outfurther below. Thus the invention provides a cell comprising the vectorsof the present invention.

In an aspect, the present invention provides a method for preparing aterpene synthase comprising the steps of: culturing a host organismand/or cell modified to contain at least one nucleic acid sequence underconditions that provide for the expression of said encoded terpenesynthase, wherein said at least one nucleic acid is a nucleic acidaccording to the invention. The terpene synthase can then optionally bepurified. Examples of such production methods are provided in, forexample, Examples 4, 5 and 12.

For example, the method of producing a terpene synthase may comprise thesteps of:

(a) selecting a host organism and/or cell which does not express anucleic acid having a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3or SEQ ID NO: 5;

(b) transforming the organism with a nucleic acid molecule having asequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5; and

(c) culturing the organism under conditions conducive to the productionof the terpene synthase encoded by said nucleic acid.

The present invention also provides a method of producing a terpenesynthase, the method comprising the steps of:

(a) selecting a host organism and/or cell which does express a nucleicacid molecule having a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3or SEQ ID NO: 5;

(b) transforming the organism to express the nucleic acid moleculehaving a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO:5 in higher quantity; and

(c) culturing the organism under conditions conducive to the productionof the terpene synthase encoded by said nucleic acid.

There is also provided a method of making a terpene, comprisingcontacting an acyclic pyrophosphate terpene precursor with the terpenesynthase of the present invention, wherein the terpene synthase isheterologously expressed in a cell; the acyclic pyrophosphate terpeneprecursor is expressed in the same cell as the terpene synthase; and thestep of contacting the acyclic pyrophosphate terpene precursor occurs inthe cell.

There is also provided a method of making at least one terpenecomprising: cultivating a cell under conditions conducive to theproduction of a terpene, wherein the cell heterologously expresses theterpene synthase of the invention; and the cell expresses an acyclicpyrophosphate terpene precursor.

The terpene synthases so produced may optionally be further isolated.

The acyclic pyrophosphate terpene precursor may be selected from amonggeranyl-pyrophosphate (GPP), farnesyl-diphosphate (FPP) andgeranylgeranyl-pyrophosphate (GGPP).

Furthermore, the terpene is selected from among a sesquiterpene, aditerpene and a monoterpene, such as a bicyclic or tricyclicsesquiterpene, including preferably the terpene is selected from amongα-santalene, α-trans-bergamotene, epi-β-santalene, β-santalene,γ-curcumene, β-bisabolene, β-curcumene, α-curcumene,trans-trans-farnesol, α-pinene, camphene, limonene and α-terpinolene.

The terpene may preferably be selected from among (+)-epi-β-santalene,(−)-β-santalene, (+)-β-santalene, (+)-α-santalene, and (−)-α-santalene.

In a further aspect, two or more terpenes may be produced.

When the terpene is α-santalene, the amount of α-santalene produced ispreferably at least or at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80% or more of thetotal amount of terpene(s) that is produced.

When the terpene is β-santalene, the amount of β-santalene produced ispreferably at least or at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80% or more of thetotal amount of terpene(s) that is produced.

Most preferably, at least four terpenes are produced; the four terpenesinclude α-santalene, α-trans-bergamotene, epi-β-santalene andβ-santalene; and α-santalene, α-trans-bergamotene, epi-β-santalene andβ-santalene are produced with the following proportions relative to eachother: α-santalene (38.0%), α-trans-bergamotene (12.1%), epi-β-santalene(4.7%) and β-santalene (45.2%).

The terpenes so produced may be converted to an alcohol, for example analcohol selected from among α-santalol, β-santalol, α-trans-bergamotoland epi-β-santalol. Such alcohols may, upon further processing, have thefollowing proportions relative to each other: α-santalol (25-65%),α-trans-bergamotol (1-20%), epi-β-santalol (1-15%) and β-santalol(20-50%); or more preferably, α-santalol (59.28%), α-trans-bergamotol(7.32%), epi-β-santalol (3.45%) and β-santalol (29.0%).

In a further aspect, the present invention provides a recombinant hostorganism and/or cell transformed to harbour the nucleic acid of theinvention. The host organism may be a unicellular or a multi-cellularorganism, but is non-human. The host may, for example, be a cell of amulticellular organism. Preferably, the host organism heterologouslycomprises a nucleic acid of the invention. The host cells of the presentinvention may thus be prokaryotic, bacterial, or eukaryotic cells (e.g.,yeast cells or plant cells).

As used herein, heterologous nucleic acid is nucleic acid that is notnormally produced in vivo by the cell in which it is expressed, or thatis normally produced by the cell but is at a different locus orexpressed differently or that mediates or encodes mediators that alterexpression of endogenous nucleic acid, such as DNA, by affectingtranscription, translation, or other regulatable biochemical processes.Heterologous nucleic acid is generally not endogenous to the cell intowhich it is introduced, but has been obtained from another cell orprepared synthetically. Heterologous nucleic acid also can beendogenous, but is nucleic acid that is expressed from a different locusor altered in its expression. Generally, although not necessarily, suchnucleic acid encodes RNA and proteins that are not normally produced bythe cell or in the same way in the cell in which it is expressed.Heterologous nucleic acid, such as DNA, also can be referred to asforeign nucleic acid, such as DNA. Thus, heterologous nucleic acid orforeign nucleic acid includes a nucleic acid molecule not present in theexact orientation or position as the counterpart nucleic acid molecule,such as DNA, is found in a genome. It also can refer to a nucleic acidmolecule from another organism or species (i.e., exogenous).

Heterologous expression refers to the expression in a host cell of apolypeptide encoded by heterologous nucleic acid that has beenintroduced, such as by transformation, electroporation, transduction, orany other means, into the host cell.

Preferably, the host organism is a bacterium, for example E. coli.

Further preferred host organisms include fungi, preferably yeasts, mostpreferably Saccharomyces cerevisiae. Other suitable host cells includethe BTR yeast strain described in U.S. Pat. No. 6,531,303.

Suitable host organisms for expression of polypeptides of the inventionmay alternatively be a higher eukaryotic cell, preferably a plant orplant cell. Preferably, the plant is a species belonging to the familyof the Solaniaceae or Lamiaceae, more preferably the genus of Nicotiana;or alternatively, a member of the genus Catharanthus such asCatharanthus vinca. The host organism may alternatively be a species ofthe Santalum genus, such as S. album or S. spicatum.

In an aspect, the present invention provides a recombinant host organismor cell expressing the polypeptide of the present invention. Preferably,the host organism is transformed to express the polypeptide in a higherquantity than in the same organism not so transformed.

The term “transformed” refers to the fact that the host was subjected togenetic engineering to comprise one, two, or more copies of any of thenucleic acids of the invention. Preferably, the host heterologouslyexpresses the nucleic acids and/or polypeptides of the invention.

Accordingly, in an embodiment, the present invention provides atransformed organism in which the polypeptide of the invention isexpressed in a higher quantity than in the same organism not sotransformed.

There are several methods known in the art for the creation oftransgenic, recombinant host organisms or cells such as plants, yeasts,bacteria, or cell cultures of higher eukaryotic organisms. For example,appropriate cloning and expression vectors for use with bacterial,fungal, yeast, and mammalian cellular hosts are described, for example,in Pouwels et al., “Cloning Vectors: A Laboratory Manual,” Elsevier, NewYork (1985); and Sambrook et al., cited above. Cloning and expressionvectors for higher plants and/or plant cells in particular are availableto the skilled person (see, for example, Schardl et al., Gene 61:1-11(1987)).

Methods for transforming host-organisms, for example, producingtransgenic plants, modifying host organisms or cells to harbortransgenic nucleic acids, such as those of the present invention, arefamiliar to the skilled person. For the creation of transgenic plants,for example, current methods include: electroporation of plantprotoplasts, liposome-mediated transformation, agrobacterium-mediatedtransformation, polyethylene-glycol-mediated transformation, particlebombardment, microinjection of plant cells, and transformation usingviruses.

In one embodiment, transformed DNA is integrated into a chromosome of anon-human host organism and/or cell such that a stable recombinantsystem results. Any chromosomal integration method known in the art maybe used in the practice of the invention, including, but not limited to,recombinase-mediated cassette exchange (RMCE), viral site-specificchromosomal insertion, adenovirus, homologous recombination byhost-mediated processes and pronuclear injection.

The host cell of the present invention may preferably naturally producethe FPP substrate for the terpene synthase. Alternatively, the hostcells can be engineered by, for example, transformation using aheterologous FPP gene, to produce FPP if they do not naturally producesuch a compound. Further, host cells can be engineered to produce moreFPP that they would naturally, thereby providing larger amounts of thesubstate than the terpene synthase of the present invention uses (see,for example, U.S. Pat. No. 6,531,303 or WO 2009/109597).

It is anticipated that the sesquiterpene products of the enzymes of thepresent invention may be further processed by hydroxylation at the C₁₂position to form santalols and bergamotols. For example, the α-santalenewould be further processed to yield α-santalol, α-trans-bergamotenewould yield α-trans-bergamotol, epi-β-santalene would yieldepi-β-santalol and β-santalene would yield β-santalol. The SaSSy, SauSSyor SspiSSy terpene synthases of the present invention are preferablyable to synthesise α-santalene, α-trans-bergamotene, epi-β-santalene andβ-santalene in proportions relative to each other such that, on furtherprocessing of the compounds to the equivalent alcohols, the yield ofsantalols and bergamotols would be similar to that of the natural oilof, for example, S. album sandalwood.

Generally, sandalwood oil from S. album is composed of compounds asshown in Table 1, with the given proportions.

TABLE 1 Example of the composition of natural sandalwood oil (from S.album) Compound % composition by GC α-santalene 1.26 β-santalene 0.22E-α-bergamotene 1.28 epi-β-santalene 1.81 γ-curcumene 0.049 β-bisabolene0.029 β-curcumene 0.226 α-curcumene 0.180 α-bisabolol 0.01 Z-α-santalol51.48 Z-α-trans-bergamotol 6.36 Z-epi-β-santalol 3.80 Z-β-santalol 25.0E-β-santalol 0.211 Z-lanceol 1.76 Z-nuciferol 1.30

It can be determined from Table 1 above that the proportions ofα-santalol, α-trans-bergamotol, epi-β-santalol and β-santalol relativeto each other (minus other oil components) are: α-santalol (59.28%),α-trans-bergamotol (7.32%), epi-β-santalol (4.35%) and β-santalol(29.0%).

The terpene synthases of the present invention preferably synthesizeα-santalene, α-trans-bergamotene, epi-β-santalene and β-santalene suchthat, on further processing, the compounds yield the respective alcoholsin the following proportions relative to each other: α-santalol(25-65%), α-trans-bergamotol (1-20%), epi-β-santalol (1-15%) andβ-santalol (20-50%).

Most preferably, the terpene synthases of the present inventionsynthesise the sesquiterpene compounds such that, on further processing,the compounds yield the respective alcohols in the following relativeproportions: α-santalol (34.7%), α-trans-bergamotol (11.1%),epi-β-santalol (4.3%) and β-santalol (41.3%). The remaining 8.6% ofcompounds are generally made up of a range of other compounds in smallamounts. If the proportion of the other minor compounds is eliminated,the remaining four major compounds, on further processing to yield therespective alcohols, are preferably generated in the followingproportions relative to each other: α-santalene (38.0%),α-trans-bergamotene (12.1%), epi-β-santalene (4.7%) and β-santalene(45.2%).

The relative proportions of the four major components in the oilproduced using an isolated SaSSy terpene synthase and converted to therespective santalols was determined by both GC-MS and GC-FID. It isknown that GC-MS tends to bias some compounds based on ease ofionization. However, both methods indicate that the relative compositionof the products of the terpene synthases of the present invention arereflective of the composition of the respective alcohols in sandalwoodoil (see Table 2).

TABLE 2 Relative % Relative % Relative % Relative % compositioncomposition composition composition Relative % in oil in oil in oil inoil composition produced produced produced produced in native oil bySaSSy by SaSSy by SauSSy by SspiSSy Compound (GC-MS) (GC-MS) (GC-FID)(GC-FID) (GC-FID) α-santalol 59.28 38.0 45.3 51.3 47.3 β-santalol 29.045.2 31.5 27.4 26.3 E-α-bergamotol 7.32 12.1 16.0 14.5 19.2epi-β-santalol 4.35 4.7 4.7 4.8 4.2

The further processing may be carried out by any means known to theskilled person, such as use of an appropriate cytochrome P450 enzyme, orchemical reactions such as alkaline metalation, borylation andoxidation, to yield the correct cis alcohols. Cytochrome P450 technologyis established for other sesquiterpenoids, most notably artemisininproduction.

The nucleic acid probes of the present invention may be used to selectsuitable trees for breeding programs or strain improvement programs inrelation to S. album or other members of the Santalum genus. It is knownthat not all S. austrocaledonicum, S. spicatum and S. album treesproduce sandalwood oil in equivalent quantities, and the relativeproportion of terpenes in the oil may be the subject of variationbetween trees, particularly in relation to the control, expressiontranscription and/or translation of the nucleic acids of the SaSSy,SauSSy or SspiSSy genes.

Therefore, the nucleic acid probes of the present invention may be usedto determine which trees of a given set are expressing SaSSy, SauSSy orSspiSSy genes at high levels, using methods described herein and/orthose well known to the skilled addressee. Alternatively, the level ofexpression of SaSSy, SauSSy or SspiSSy amino acids may be determined toassess which trees are producing large amounts of desirable terpenes.Preferably, probes may be used to detect trees that express a gene for aterpene synthase that produces one or more of the following terpenes:α-santalene, α-trans-bergamotene, epi-β-santalene, β-santalene andtrans-trans-farnesol.

Alternatively, the polypeptides or polypeptide fragments of the presentinvention may be used to generate antibodies against specific regions ofthe SaSSy, SauSSy or SspiSSy genes. These antibodies may then be used toscreen samples from trees to determine which trees are expressing thesynthase genes, and the level of that expression. Again, such methods ofdetection may be used to select trees that produce large amounts ofdesirable terpenes, or desirable proportions of different terpenes.

The invention thus provides a method for detecting the presence of aterpene synthase in a sample, comprising the steps of:

(a) contacting a sample suspected of containing terpene synthase of theinvention with an antibody that specifically binds to the terpenesynthase under conditions which allow for the formation of reactioncomplexes comprising the antibody and the terpene synthase; and

(b) detecting the formation of reaction complexes comprising theantibody and the terpene synthase in the sample, wherein detection ofthe formation of reaction complexes indicates the presence of theterpene synthase amino acid sequence in the sample.

The method may comprise the further step of evaluating the amount ofreaction complexes formed, thereby determining the amount of terpenesynthase in the biological sample.

Preferably, the antibody used in these methods is derived from anaffinity-purified polyclonal antibody, and more preferably a mAb. Inaddition, it is preferable that the antibody molecules used herein be inthe form of Fab, Fab′, F(ab′)₂ or F(v) portions or whole antibodymolecules.

Particularly preferred methods for detecting the terpene synthase genesof the invention based on the above methods include enzyme-linkedimmunosorbent assays, radioimmunoassays, immunoradiometric assays andimmunoenzymatic assays, including competitive and sandwich assays usingmonoclonal and/or polyclonal antibodies.

In each instance, the amino acid sequences of the present invention formcomplexes with one or more antibody(ies) or binding partners and onemember of the complex is labelled with a detectable label. The fact thata complex has formed and, if desired, the amount thereof, can bedetermined by known methods applicable to the detection of labels.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals that fluoresce when exposed to ultravioletlight, and others.

A number of fluorescent materials are known and can be utilized aslabels. These include, for example, fluorescein, rhodamine and auramine.A particular detecting material is anti-rabbit antibody prepared ingoats and conjugated with fluorescein through an isothiocyanate.

The SaSSy, SauSSy or SspiSSy amino acid sequences or their bindingpartners can also be labelled with a radioactive element or with anenzyme. The radioactive label can be detected by any of the currentlyavailable counting procedures.

Enzyme labels are likewise useful, and can be detected by any of thepresently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques. Theenzyme is conjugated to the selected particle by reaction with bridgingmolecules such as carbodiimides, diisocyanates, glutaraldehyde and thelike. Many enzymes which can be used in these procedures are known andcan be utilized. The preferred enzymes are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090;3,850,752; and 4,016,043 are referred to by way of example for theirdisclosure of alternate labelling material and methods.

Other preferred methods for detecting the terpene synthase genes andproteins of the invention based on the above methods include selectivereaction monitoring or multiple reaction monitoring, as discussed inZulak, K. G., Lippert, D. N., Kuzyk, M., Domanski, D., Chou, T.,Borchers, C. H. and J. Bohlmann, “Targeted proteomics using selectedreaction monitoring (SRM) reveals the induction of specific terpenesynthases in a multi-level study of methyl jasmonate treated Norwayspruce (Picea abies),” The Plant Journal 60:1015-1030 (2009).

The present invention further provides methods for detecting thepresence of a terpene synthase nucleic acid molecule in a biologicalsample, which comprise the steps of:

(a) bringing the biological sample into contact with a polynucleotideprobe or primer comprising a terpene synthase polynucleotide of theinvention under suitable hybridising conditions; and

(b) detecting any duplex formed between the probe or primer and nucleicacid sequences in the sample.

According to one embodiment of the invention, detection of the SaSSy,SauSSy or SspiSSy genes may be accomplished by directly amplifying theterpene synthase polynucleotide sequences from a biological sample,using known techniques, and then detecting the presence of the SaSSy,SauSSy or SspiSSy polynucleotide sequences.

The present invention thus also relates to a method for the detection ofa terpene synthase in a biological sample, comprising:

(a) amplifying the nucleic acid molecule with at least one primer oroligonucleotide as defined above; and

(b) detecting the amplified nucleic acid molecules.

Preferably, the nucleic acid is extracted and/or purified (e.g., from atissue sample) prior to amplification.

The present invention also relates to a method for the detection ofSaSSy, SauSSy or SspiSSy nucleic acids present in a biological sample,comprising:

(a) hybridizing the nucleic acids of the biological sample atappropriate conditions with one or more probes as defined above;

(b) washing under appropriate conditions; and

(c) detecting the hybrids formed.

Preferably, the hybridizing conditions are denatured conditions.

Preferably, the nucleic acid is extracted and/or purified (e.g., from atissue sample) prior to hybridisation. More preferably, the nucleic acidsample is amplified with at least one primer as defined above, afterextraction or at least prior to hybridisation. Preferably, said probesare attached to a solid substrate or detected in a liquid phase byphotometric or fluorogenic detection or by other methods ofvisualisation such as by agarose gel electrophoresis.

The present invention also relates to a method as defined above, whereinsaid nucleic acids are labelled during or after amplification.

Suitable assay methods for purposes of the present invention to detecthybrids formed between the oligonucleotide probes and the nucleic acidsequences in a sample may comprise any of the assay formats known in theart, such as the conventional dot-blot format, sandwich hybridization orreverse hybridization. For example, the detection can be accomplishedusing a dot blot format, the unlabelled amplified sample being bound toa membrane, the membrane being incorporated with at least one labelledprobe under suitable hybridization and wash conditions, and the presenceof bound probe being monitored.

An alternative and preferred method is a “reverse” dot-blot format, inwhich the amplified sequence contains a label. In this format, theunlabelled oligonucleotide probes are bound to a solid support andexposed to the labelled sample under appropriate stringent hybridizationand subsequent washing conditions. It is to be understood that also anyother assay method which relies on the formation of a hybrid between thenucleic acids of the sample and the oligonucleotide probes according tothe present invention may be used.

In one form of the invention, the target nucleic acid sequence isamplified by PCR and then detected using any of the specific methodsmentioned above. Other useful diagnostic techniques for detecting thepresence of SaSSy, SauSSy or SspiSSy polynucleotide sequences include,but are not limited to: 1) allele-specific PCR; 2) single strandedconformation analysis; 3) denaturing gradient gel electrophoresis; 4)RNase protection assays; 5) the use of proteins which recognizenucleotide mismatches, such as the E. coli mutS protein; 6)allele-specific oligonucleotides; and 7) fluorescent in situhybridisation.

In addition to the above methods, SaSSy, SauSSy or SspiSSypolynucleotide sequences may be detected using conventional probetechnology. When probes are used to detect the presence of the SaSSy,SauSSy or SspiSSy polynucleotide sequences, the biological sample to beanalysed, such as woody tissue particularly heartwood tissue, may betreated, if desired, to extract the nucleic acids. The samplepolynucleotide sequences may be prepared in various ways to facilitatedetection of the target sequence, e.g., denaturation, restrictiondigestion, electrophoresis or dot blotting. The targeted region of thesample polynucleotide sequence usually must be at least partiallysingle-stranded to form hybrids with the targeting sequence of theprobe. Denaturation of the target sequence will probably be required andcan be carried out by various techniques known in the art.

Sample polynucleotide sequences and probes are incubated underconditions that promote stable hybrid formation of the target sequencein the probe with the putative SaSSy, SauSSy or SspiSSy polynucleotidesequence in the sample. Preferably, high stringency conditions are usedin order to prevent false positives.

Detection, if any, of the resulting hybrid is usually accomplished bythe use of labelled probes. Alternatively, the probe may be unlabelled,but may be detectable by specific binding with a ligand that islabelled, either directly or indirectly. Suitable labels and methods forlabelling probes and ligands are known in the art, and include, forexample, radioactive labels which may be incorporated by known methods(e.g., nick translation, random priming or kinasing), biotin,fluorescent groups, chemiluminescent groups (e.g., dioxetanes,particularly triggered dioxetanes), enzymes, antibodies and the like.Variations of this basic scheme are known in the art, and include thosevariations that facilitate separation of the hybrids to be detected fromextraneous materials and/or that amplify the signal from the labelledmoiety.

Preferably, the probe is labelled. More preferably, the probe isradiolabelled or fluorescent- or enzyme-labelled.

Once suitable trees have been identified, they may be used in selectivebreeding programs, or simply identified as trees which may be harvestedfor sandalwood oil production.

In a still further aspect, the present invention provides processesand/or methods for making terpenoids.

Accordingly, the present invention provides a method of making at leastone terpenoid comprising:

(a) contacting at least one acyclic pyrophosphate terpene precursor withat least one polypeptide of the invention or encoded by any of thenucleic acids of the invention; and

(b) optionally, isolating at least one terpenoid produced in step (a).Furthermore, the present invention provides a method of making at leastone terpenoid comprising:

(a) cultivating a non-human organism transformed to express orincreasingly express a polypeptide of the invention under conditionsconducive to the production of terpenoids; and

(b) optionally, isolating at least one terpenoid from the non-humanorganism.

According to a preferred embodiment, the method further comprises thestep of: transforming a non-human organism with a recombinant nucleicacid to express or increasingly express a polypeptide of the invention,before step (a).

Preferably, the at least one terpenoid is selected from the following:α-santalene, α-trans-bergamotene, epi-β-santalene and β-santalene.

The method of making at least one terpenoid comprises the step ofcontacting at least one acyclic pyrophosphate terpene precursor with atleast one polypeptide of the invention. For example, polypeptides asobtained in the above methods for producing terpene synthases may beused. Such polypeptides may be extracted from host organisms expressingthe nucleic acids of the invention according to standard protein orenzyme extraction technologies. If the host organism is a unicellularorganism or cell releasing the polypeptide of the invention into theculture medium, the polypeptide may be collected from the culturemedium, for example, by centrifugation, optionally followed by washingsteps and resuspension in suitable buffer solutions.

If the host organism is a plant or a unicellular organism or cellaccumulating the polypeptide of the invention within the cell, thepolypeptide may be obtained by disruption or lysis of the cells andextracting the polypeptide from the cell lysate.

The isolated polypeptide may then be suspended in a buffer solution atoptimal pH and temperature. If adequate, salts, BSA and other kinds ofenzymatic co-factors may be added in order to optimize enzyme activity.

The terpene precursor may be added to a polypeptide suspension orsolution, followed by incubation at optimal temperature, for example300° C. After incubation, the terpenoid compound may be isolated fromthe incubated solution by standard isolation procedures, such as solventextraction and distillation, preferably after removal of polypeptidesfrom the solution.

In a step of the process for making at least one terpenoid compound, thehost organism or cell is cultivated under conditions conducive to theproduction of terpenoids. Accordingly, if the host is a transgenicplant, optimal growing conditions are provided, such as optimal light,water and nutrient conditions, for example. If the host is a unicellularorganism, conditions conducive to the production of the terpenoid maycomprise addition of suitable cofactors to the culture medium of thehost. In addition, a culture medium may be selected which proves tomaximize terpenoid synthesis. External factors such as optimized pH andtemperature are usually also conducive to terpenoid production in agiven expression system.

Other factors such as elicitors can also be used to upregulatetranscription of certain genes, in particular terpenes, which can beassociated with plant defense mechanisms. Elicitors may therefore beused to upregulate the transcription of the terpene synthase gene of thepresent invention, in sandalwood or other host organisms, includinghosts trees. A range of elicitors are well known to those skilled in theart and can be readily purchased. Preferably, the elicitor chosen is onethat upregulates the expression of the terpene synthase gene of thepresent invention in the specific environment in which the gene isprovided. Examples of suitable elicitors include methyl jasmonate andsalicylic acid. The terpene synthase gene may further provide a means toassess whether upregulation of transcription has occurred followingtreatment of, for example, a sandalwood tree, with elicitors. Suchassessment may further include the measurement of oil levels in suchtrees to determine if the elicitor applied has an upregulating effect.

In a further embodiment of this invention, kits may be prepared todetermine the presence or absence of a SaSSy, SauSSy or SspiSSy gene insandalwood trees and/or the activity of the gene.

In accordance with the testing techniques discussed above, one class ofsuch kits for the detection of the SaSSy, SauSSy or SspiSSy proteinswill contain at least a labelled SaSSy, SauSSy or SspiSSy amino acidsequence binding partner, for instance an antibody specific thereto, anddirections depending upon the method selected, e.g., “competitive,”“sandwich,” “DASP,” and the like. The kits may also contain peripheralreagents such as buffers, stabilizers, etc.

Accordingly, a test kit may be prepared for the demonstration of thepresence of the SaSSy, SauSSy or SspiSSy enzymes comprising:

(a) a predetermined amount of at least one labelled immunochemicallyreactive component obtained by the direct or indirect attachment of aSaSSy, SauSSy and SspiSSy amino acid sequence specific binding partnerto a detectable label;

(b) other reagents; and

(c) directions for use of said kit.

The labelled binding partner (such as an antibody) may be generallybound to a solid phase.

The invention also provides kits for detecting SaSSy, SauSSy or SspiSSynucleic acid sequences.

Accordingly, the invention provides a kit for demonstrating the presenceof SaSSy, SauSSy or SspiSSy nucleic acid sequences comprising:

(a) a predetermined amount of at least one labelled nucleic acidsequence derived from the SaSSy, SauSSy or SspiSSy gene sequence;

(b) other reagents; and

(c) directions for use of said kit.

For example, the polynucleotide sequence may be one or more primers,such as those exemplified above, and the instructions for use may beinstructions to perform PCR on RNA or DNA extracted from a tissue samplefrom a subject.

In another aspect, the invention provides a kit for demonstrating thepresence of a terpene synthase comprising:

(a) a predetermined amount of at least one ligand that binds to theterpene synthase of the present invention, wherein the ligand comprisesa detectable label;

(b) other reagents; and

(c) directions for use of said kit.

In a further aspect there is provided a kit for demonstrating thepresence of nucleic acid molecules encoding a terpene synthase,comprising:

(a) a predetermined amount of at least one labelled nucleic acidmolecule or primer of the present invention;

(b) other reagents; and

(c) directions for use of said kit.

The above kits may use samples from a sandalwood tree. The method may beperformed on at least two samples from two different sandalwood trees;and the amount of terpene synthase or nucleic acid encoding terpenesynthase in each sample is determined; then the sandalwood tree fromwhich the sample with the most amount of a terpene synthase or a nucleicacid encoding a terpene synthase was derived, is selected; and theselected sandalwood tree is selectively bred or harvested for sandalwoodoil.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in the specification, individually or collectively andany and all combinations or any two or more of the steps or features.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally equivalent products, compositions andmethods are clearly within the scope of the invention as describedherein.

The entire disclosures of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference. Noadmission is made that any of the references constitute prior art or arepart of the common general knowledge of those working in the field towhich this invention relates.

As used herein, the terms “derived” and “derived from” shall be taken toindicate that a specific integer may be obtained from a particularsource, albeit not necessarily directly from that source.

As used herein, the singular forms “a,” “an” and “the” include pluralreferences unless the context clearly dictates otherwise. Thus, forexample, reference to a terpene synthase that catalyzes the formation ofa terpene includes synthases that catalyze the productions of one or aplurality of terpenes.

Throughout this specification, unless the context requires otherwise,the word “comprise,” or variations such as “comprises” or “comprising,”will be understood to imply the inclusion of a stated integer or groupof integers, but not the exclusion of any other integer or group ofintegers.

Other than in the operating example, or where otherwise indicated, allnumbers expressing quantities of ingredients, reaction conditions, andso forth used in the specification and claims are to be understood asbeing modified in all instances by the term “about.” Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thespecification and claims are approximations that may vary depending uponthe desired properties sought to be obtained by the present invention.Hence “about 80%” means “about 80%” and also “80%.” At the very least,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

Other definitions for selected terms used herein may be found within thedetailed description of the invention and apply throughout. Unlessotherwise defined, all other scientific and technical terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the invention belongs.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these methods in no way serve to limit the true scope ofthis invention, but rather are presented for illustrative purposes.

EXAMPLES

All reagents, solvents, antibiotics and precursor chemicals werepurchased from commercial sources. Farnesyl diphosphate and geranyldiphosphate were from Sigma (St. Louis, Mo., USA). Restrictionendonucleases and T4 DNA ligase were from New England Biolabs (Ipswich,Mass.).

Example 1 Plant Material Collection and RNA Extraction

Several holes were drilled into one mature Santalum album tree growingin the FPC Kununurra arboreturn, Kununurra, Wash. Wood shavings from thetransition zone of the xylem were collected and frozen immediately inliquid nitrogen and transported to UWA, Perth.

RNA was extracted using a modified protocol (Kolosova, N., et al.,“Isolation of high-quality RNA from gymnosperm and angiosperm trees,”BioTechniques 36:821-824 (2004)). Wood shavings (50 g) were ground inliquid nitrogen and added to RNA extraction buffer (200 mM Tris-HCl, pH8.5, 1.5% lithium dodecyl sulphate, 300 mM LiCl, 10 mM EDTA, 1% w/vsodium deoxycholate, 1% w/v Tergitol Nonidet® P-40). 5 mM thiourea, 1 mMaurintricarboxylic acid, 10 mM dithiothreitol, and 2% (w/v)polyvinylpolypyrrolidone (PVPP) were added just prior to use. Allsolutions were prepared from DEPC treated and/or autoclaved water.Several tubes were combined at the TE/NaCl resuspension step toconcentrate the RNA sample. After precipitation, RNA was stored at −80°C. until transported to UBC Vancouver, Canada for cDNA libraryconstruction.

Example 2 cDNA Synthesis and Library Construction

1.4 μg of S. album xylem total RNA was reverse transcribed withSuperScript III reverse transcriptase (Invitrogen) at 42° C. for 1 hourusing the Clontech oligo dT primer and SMART 5′ oligo. cDNA wasamplified using the M1 primer supplied in the kit.

The amplified cDNA was then digested with Sfi1 restriction enzyme (NewEngland Biolabs). The resulting mixture was passed through a Chromaspinsize exclusion column, eluting the largest sized fragments first. cDNAwas collected in 200 mL aliquots and those which had the desired highmolecular weight range were combined and eluted on a Qiagen MinElutespin column.

The digested cDNA fragments were then cloned into pre-cut pDNR-LIBvector and transformed by electroporation into 25 μL of phage resistantelectrocompetent E. coli cells. The cells were shaken at 37° C. for 1 hin SOC media before being mixed with glycerol and stored at −80° C.Aliquots were plated onto agar containing chloramphenicol at 30 mg/mL.cDNA library was titrated to approximately 3×106 colony forming unitsper mL. The library was sent to the Genome Sciences Centre, VancouverCanada for 5′ and 3′ sequencing.

Example 3 Identification of Terpene Synthase Genes from an S. albumXylem EST Library

Reads were assembled using the CAP3 program with default settings. TheEST library was compared by BLAST searching to the NCBI database forgenes homologous to previously published terpene synthase (TPS) andcytochrome P450 gene sequences. Several candidate genes were identified,including one full length gene with modest identity to limonene synthasefrom Ricinus communis and linalool synthase Backhousia citriodora. About12 putative cytochrome P450 oxidase enzymes were also discovered, someof which were full length.

Example 4 Bacterial Expression

Colonies producing a positive hit to known terpene synthase (TPS) geneswere plasmid purified to yield pDNR-LIB bound full length cDNAs. Onegene, labelled SaSSy, was selected. Alignment of the deduced amino acidsequence of SaSSy with other angiosperm genes are shown in FIG. 6 andalignment of the deduced amino acid sequence of SaSSy, SauSSy andSspiSSy with other angiosperm terpene synthase genes are shown in FIG.12.

Two versions of the SaSSy gene were located, differing only at position143, with clone P143 having a proline residue at position 143 and cloneS143 having a serine residue at that location. The two proteins producedfrom these clones were later found to have identical activity,indicating that switching one polar residue with another at position 143has little to no effect on catalysis.

SaSSy features motifs typical of the monoterpene synthase gene TPS-bsubfamily (Bohlmann, J., et al., “Plant terpenoid synthases: molecularbiology and phylogenetic analysis,” Proc. Natl. Acad. Sci. U.S.A.95:4126-4133 (1998)), including the aspartate-rich DDxxD metal ionbinding site (positions 321-325) and the RRX₈W motif (positions 32-42),which is implicated in diphosphate group migration (Williams, D. C., etal., “Truncation of limonene synthase preprotein provides a fully active‘pseudomature’ form of this monoterpene cyclase and reveals the functionof the amino-terminal arginine pair,” Biochem. 37:12213-12220 (1998)).

The two full length SaSSy gene sequences located above in Example 4(P143 and S143) were cloned into pET28b (+) expression vectors (Novagen,San Diego, Calif.) with a C-terminal histidine tag. The vectors were cutwith NcoI and XhoI, to create overhangs suitable for ligation intosimilarly digested cDNA. The circular plasmids, which contained thesesquiterpene synthase gene in frame with a Ni-affinity tag (His₆) werefirst transformed into chemically competent cells and grown on LB plateswith kanamycin (50 μg/mL).

Transformants were grown on selective LB plates (kanamycin 50 μg/mL),and the DNA extracted via plasmid preparation (Invitrogen). pET28b (+)vectors containing the insert were sequence verified and transformedinto chemically competent C41 E. coli cells (Avidis, Saint-Beauzire,France) containing the pRARE 2 plasmid isolated from Rosetta 2 competentcells (Novagen). Colonies were grown on LB plates containing kanamycinand chloramphenicol (50 μg/mL).

Independent colonies were picked and grown in a shaker overnight at 37°C. in 5 mL of LB with the same antibiotics. 2 mL of this culture wasadded to 4 lots of 100 mL of TB containing kanamycin andchloramphenicol. The cell suspension was grown for 4 hours with shakingat 37° C. until the OD₆₀₀=0.8. Isopropyl-β-D-thiogalacto-pyranoside(IPTG) was added to a final concentration of 0.2 mM and the mixture wasshaken overnight at 16° C. Cell suspension was harvested bycentrifugation at 4° C. and the cell pellet frozen at −80° C. for futureuse.

Cell pellets (˜7 g) were resuspended in 5 mL of lysis buffer (25 mglysozyme, 0.25 mg DNAse, 0.25 mg RNAse, 19.3 mg DTT, 5 μL proteaseinhibitor cocktail [PMSF and thiourea] and 250 μL of 100 mM MgCl₂).While on ice, the mixture was stirred thoroughly with a glass rod for 30min. Lysate was then sonicated on ice using a Bronson ultrasonic probeuntil translucent. The mixture was centrifuged at 20,000×g at 4° C. for30 min. Supernatant was decanted and stored on ice.

Example 5 Protein Purification

The cleared lysate supernatant (˜3 mL) was sequentially loaded ontoHisTrap Spin columns (GE Healthcare) and spun at 1000 rpm for 2 min at4° C. After washing with fresh binding buffer the protein was elutedtwice with 300 μL of elution buffer (20 mM HEPES pH 7.5, 150 mM NaCl and350 mM imidazole) to a final volume of 600 pt.

The eluates from each of the two SaSSy terpene synthase variants (P143and S143) solutions were desalted on a 2 mL sephadex desalting column(BioRad, Hercules Calif., USA) into elution buffer which lackedimidazole.

The eluent was analysed by spectrophotometry to determine approximateprotein concentrations. Final concentration was ˜8 mg/mL. Thesesolutions were used for assays. Aliquots of the purified proteins weremixed with 80% glycerol (1:1) and frozen in liquid N₂. These were storedat −80° C. until use. Proteins were analysed using SDS-PAGE, westernblotting and immunolabelling with His₆ antibody (Sigma). Antibody wasvisualised by the NBT/BCIP assay (Roche, USA). The recombinant proteinfor each of P143 and S143 was confirmed to be of the expected size of˜66 kDa.

Example 6 Enzyme Assays

Enzyme assays for both recombinant isoenzyme proteins P143 and S143 weredone in duplicate using GC vials as per O'Maille, P. E., et al. (“Asingle-vial analytical and quantitative gas chromatography-massspectrometry assay for terpene synthases,” Anal. Biochem. 335:210-217(2004)). For each assay, 20 μL of protein (8 mg/mL) was added to 450 μLof reaction buffer containing 25 mM HEPES, 10% glycerol, 5 mM DTT and 10mM of Mg²⁺. 20 μL of substrates FPP and GPP (˜1 mg/mL) were added andthe mixture gently shaken. Final enzyme concentration was 0.3 mg/mL.Vials were overlaid with 500 μL of pentane to trap volatile products andincubated at 30° C. for 2 h.

Example 7 GC-MS Analysis and Product Identification

The product mixture of the SaSSy enzyme was analysed on an Agilent 6890GC with a 5973 MSD using helium as carrier gas. Peak identification wasdone using an HP5-MS capillary column (30 m, 0.25 mm ID, 0.25 mm filmthickness). Injector temperature was 240° C., detector temperature wasset at 250° C., injection volume was 1 μL and column flow rate was 1mL/min. Oven temperature program was as follows: yen program was heldfor 1 min at 40° C., then ramped at 7.5° C./min to 250° C. and held. Allmass spectra were detected at 70 eV in multiple ion scan mode.

The identities of products from SaSSy incubations were confirmed bycomparison of mass spectra retention times to authentic sandalwood oilin the NIST 2005 library, see publications by Adams, R. P.(“Identification of essential oil components by gas chromatography/massspectrometry,” Allured Publishing Corporation, Carol Stream, Ill.(1995)) and MassFinder (www.massfinder.com).

Enzyme assays using recombinant SaSSy enzyme with GPP as substrate inthe presence of Mn²⁺ produced a mixture of α-pinene, camphene, limoneneand α-terpinolene, along with other monoterpene products as determinedby GC-MS.

Enzyme assays using SaSSy with FPP as the substrate and Mg2+ ioncontaining buffer produced a mixture of α-santalene (34.7%),α-trans-bergamotene (11.1%), epi-β-santalene (4.3%), β-santalene (41.3%)and small amounts of several other compounds as determined by GC-MS(FIG. 2A). The mass spectrum of the four compounds produced by the SaSSyenzyme (shown in FIG. 2A) are the same as those of natural β-santaleneproduced in sandalwood trees (see Adams (1995), cited above, forcomparison profiles).

Incubation of FPP with heat-denatured enzyme resulted in no detectableproducts, indicating the enzyme was essential for product conversions.

Example 8 Comparison of Nucleic Acid Sequence

A Clustal multiple sequence alignment of terpene synthases(FB299123-125—terpene synthase from Vetiveria zizanoides (WO2006/134523); AF484125—5-epi-aristolochene synthase from Nicotianaattenuate; AB438045—linalool synthase from Backhousia citriodora;Santalene—SaSSy santalene synthase of the present invention fromSantalum album) is shown in FIG. 6. Linalool synthase from Backhousiacitriodora is the most homologous protein; however, there is a generallack of similarity between santalene and the other TPS genes. Anovverhead single line indicates approximate regions indicated by Backand Chappell (Proc. Natl. Acad. Sci. U.S.A. 93:6841-6845 (1996)) asbeing important for specific product formation, and double lines for theratio specific region of TEAS.

On the amino acid level, SaSSy santalene synthase was 56% identical tothe previously discovered monoterpene synthase SamonoTPS1 (EU798692.1),48% identical to a putative limonene synthase from Ricinus communis(EQ973796.1), and 45% identical to a linalool synthase from Backhousiacitriodora (BAG82825.1).

Example 9 Plant Material Collection and RNA Extraction

Several 25 mm holes were drilled into mature S. album, S.austrocaledonicum and S. spicatum trees growing in plantations managedby the Forest Products Commission of WA. Wood shavings from theheartwood-sapwood transition zone were collected and frozen immediatelyin liquid nitrogen. The samples were transported to the lab where RNAwas extracted using a modified protocol (N. Kolosova, B. Miller, S.Ralph, B. Ellis, C. Douglas, K. Ritland, J. Bohlmann, BioTechniques36:821-824 (2004)). Wood shavings (10 g) were ground in liquid nitrogenand added to RNA extraction buffer (200 mM Tris-HCl, pH 8.5, 1.5%lithium dodecyl sulphate, 300 mM LiCl, 10 mM EDTA, 1% w/v sodiumdeoxycholate, 1% w/v Tergitol Nonidet P-40). RNAse inhibitors (5 mMthiourea, 1 mM aurintricarboxylic acid, 10 mM dithiothreitol, and 2%polyvinylpolypyrrolidone) were added to the buffer just prior to use.All solutions were prepared from DEPC treated, autoclaved water. Wherepossible, samples from the same tree were combined at the TE/NaClresuspension step to concentrate the RNA sample. After LiClprecipitation, RNA was stored at −800° C. until transported to UBCVancouver, Canada, for cDNA library construction.

Example 10 Santalum album cDNA Library Construction

1.4 μg of S. album xylem total RNA was used to create a cDNA libraryusing the Clontech SMART-Creator library construction kit. RNA wasreverse transcribed using SuperScript III reverse transcriptase(Invitrogen). cDNA was amplified using Phusion high-fidelity DNApolymerase and the universal primer (Clontech). This was digested withSfi1 restriction endonuclease and cloned into pre-cut pDNR-LIB vector.The mixture was transformed by electroporation into 25 μL of phageresistant electrocompetent E. coli cells. The library titre wasdetermined and sent to the Genome Sciences Centre, Vancouver, Canada,for bidirectional Sanger DNA sequencing. Reads were assembled using theCAP3 program with default settings. The sequences (6000 unique reads)were compared to the GenBan database for key specialised metabolismgenes, particularly TPS genes and cytochrome P450s.

Example 11 Orthologous TPS Gene Discovery by RACE

cDNA was generated for S. austrocaledonicum and S. spicatum in the samemanner as before, except the cDNA was used directly as template for PCR.Primers based on the ORF of each gene were used for amplification (Table3). Where products could not be amplified, 5′- and 3′-RACE were used toobtain the respective UTRs for more specific primer design. SesquiTPS1gene orthologs were amplified in two rounds using a nested primerapproach.

TABLE 3  Primers Gene Forward primer Reverse primerOpen reading frame primers SaSSy ATGGATTCTTCCACCGCCCGAGCTTACTACTCCTCGCCG ACCGCC AGAGG SauSSy ATGGATTCTTCCACCGCCCGAGCTTACTACTCCTCGCCG ACCGCC AGAGG SspiSSy ATGGATTCTTCCACCGCCCGAGCTTACTACTCCTCGCCG ACCGCC AGAGGpET28b (+) cloning primers with incorporatedrestriction sites underlined SaSSy ATCCATGGATTCTTCCACATCTCGAGCTCCTCGCCGAGA CGCC GG SauSSy ATCCATGGATTCTTCCACATCTCGAGCTCCTCGCCGAGA CGCC GG SspiSSy ACGGATCCAATGGATTCTTACTCGAGTTACTACTCCTCG TCCACCGCCAC CCGAG

All products were first cloned into a high-copy storage vector (TOPOZero Blunt, Invitrogen) for sequencing before being cloned into anexpression vector. TPS genes amplified from several genotypes of S.album and S. spicatum were cloned and sequenced to examine potentialnucleotide polymorphisms in the ORFs. Genomic DNA sequences for allthree TPS genes were also cloned and sequenced for all three Santalumspecies. The same ORF primers used for successful cDNA amplificationswere used on genomic DNA extracted from the same individuals from whichRNA extractions were performed. These larger gDNA fragments (3-4 kb)were cloned and sent for sequencing (Macrogen, Korea).

Example 12 Bacterial Expression and Protein Isolation

All TPS genes were cloned into the pET28b(+) expression vector (Novagen,San Diego, Calif.) with a poly-histidine tag in frame. Depending on therestriction sites available, the His₆ tag was either N-terminal orC-terminal. Primers with appropriate restriction sites were used toamplify each gene, which was then digested, gel-purified and cloned intothe pET28b(+) vector (Table 3). All pET28b(+) constructs weresequence-verified before proceeding to recombinant protein expression.

Expression vectors containing the TPS genes were transformed intochemically competent C41 E. coli cells (Avidis, Saint-Beauzire, France)containing the pRARE 2 plasmid isolated from Rosetta 2 competent cells(Novagen). Colonies were grown on LB plates containing kanamycin andchloramphenicol (50 μg/mL). Three independent colonies were picked andgrown in a shaker overnight at 37° C. in 5 ml of LB with the sameantibiotics. This culture was used to inoculate 400 mL of selectiveTerrific Broth. The cell suspension was grown with shaking at 37° C.until the OD₆₀₀=0.8. Isopropyl-β-D-thiogalacto-pyranoside (IPTG) wasadded to a final concentration of 0.2 mM and the mixture was shakenovernight at 16° C. Cell suspension was harvested by centrifugation at4° C. and the cell pellet (˜1 g) was frozen at −80° C. for future use.

Cell pellets were resuspended in 5 mL of lysis buffer containing 1 mg/mLlysozyme, 1 mM MgCl₂, 5 mM DTT, 0.01 mg/mL DNAse1 and RNAse1, 100 μLprotease inhibitor cocktail (Sigma) and made in His-trap binding buffer(20 mM Na₂HPO₄ pH 7.4, 500 mM NaCl, 30 mM imidazole pH 7.4). On ice, thecell suspension was stirred thoroughly with a glass rod for 30 min.Lysate was then homogenized using a high pressure cell cruncher untilthe mixture was translucent, and rinsed with a further 5 mL of lysisbuffer. The lysate was centrifuged at 12000×g at 4° C. for 1.25 h beforebeing decanted. The cleared lysate (˜12 mL) was purified using Ni²⁺affinity chromatography (GE healthcare) and eluted in 600 μL of elutionbuffer (20 mM Na₂HPO₄ pH 7.4, 500 mM NaCl, 500 mM imidazole pH 7.4). Theeluted protein was desalted on a PD-10 desalting column (GE Healthcare)using 25 mM HEPES pH 7.4, 10% glycerol and 100 mM KCl. Fractions of a3.5 mL elution were collected, with the middle being the mostconcentrated. Protein concentrations were determined using a NanoDropspectrophotometer with extinction coefficients calculated by amino acidcomposition (ProtParam). SDS-PAGE was used to visualise the purifiedproteins.

Example 13 Enzyme Functional Characterisation and Kinetic Assays

Enzyme assays for all recombinant proteins were done in triplicate usingthe GC vial method described by O'Maille et al. (Anal. Biochem.335:210-2175 (2004). For enzyme assays where only product identificationwas needed, 10 μg of protein was used in a final volume of 500 μL ofreaction buffer (25 mM HEPES, 10% glycerol, 5 mM DTT and 10 mM of eitherMg²⁺ or Mn²⁺. Substrates (FPP and GPP) were added to a final reactionconcentration of 100 μM. Vials were overlaid with 500 μl, of hexane totrap volatile products and incubated at 30° C. for 2 h. Mixtures werevortexed for 1 min to extract all volatiles and the vials werecentrifuged to separate the organic layer.

For determination of steady-state enzyme kinetic constants, conditionswere as described previously except the enzyme concentration was kept at10 nM. Substrate concentrations ranged from 1 uM to 100 uM, andreactions were incubated at 30° C. for exactly 5 mins. Reactions werequenched by the addition of 500 μL 0.5 M EDTA, pH 8.0 and vortexed asabove.

The kinetic properties of the three santalene synthases are very similarwith a K_(m) around 1.65 μM for each enzyme when FPP is used assubstrate. V_(max) for assays using FPP ranged from 0.42 μM min⁻¹ forSaSSy to 0.54 μM min⁻¹ for SauSSy. K_(cat) was 0.67 min⁻¹ for SaSSy and1.66 min⁻¹ for SauSSy, which are similar to those of other publishedsynthases.

Example 14 GC-MS Analysis and Product Identification

Product mixtures were analysed by GC-MS in scan mode for productidentification. A standard containing the three santalenes andα-trans-bergamotene was prepared by flash chromatography of 2 mL of neatS. album oil over silica and eluted in hexane. A final yield of 25 mgwas resuspended in EtOAc and purity was confirmed by GC-FID withconditions described below. All mass spectra were compared to the NIST2005 library and the literature. Retention indices were determined forall compounds using an n-alkane standard and compared to the literature(R. P. Adams, “Identification of essential oil components by gaschromatography/mass spectrometry,” Allured Publishing Corporation, CarolStream, Ill. (1995)).

GC-FID was performed on a Shimadzu GC2010 with a 30 m 0.25 mm ID, 0.25μm film DB-WAX column with He as the carrier gas. Splitless injection (2μL) was used for all analyses. Conditions were as follows: injector 200°C., detector 250° C., column flow rate 1 mL/min. Oven program: 40° C.for 3 min, then 8° C./min to 180° C., held 5 min, then 10° C./min to220° C., held 10 min. Needle height was adjusted to only draw from theupper organic layer of all sample vials. GC-MS was performed on aShimadzu GC2010 with the same DB-WAX column and using He as the carriergas. Conditions were as follows: injector 200° C., MS interface 240° C.,ion source 200° C. Oven program: 40° C. for 3 min, then 8° C./min to180° C., held 5 min, then 10° C./min to 220° C., held 10 min. Solventcut time was set to 5 min. For product identification, total ionmonitoring was used, scanning from m/z 45 to m/z 300. For kineticassays, single ion monitoring of the sesquiterpene base ions m/z 91, 93and 94 were used. Likewise monoterpene base ions (m/z 69, 71 and 93)were monitored. An internal standard (isobutyl benzene, 30 μM) was addedto the hexane used to overlay each reaction. Detector response factorswere calculated based on the santalene standard prepared earlier andused to determine the product concentrations for kinetic analysis.

TABLE 4 GC-FID determination of reaction products of terpene synthasesfrom FFP in the presence of magnesium or manganese Retention SaSSy withFPP Mg SaSSy with FPP Mn time 1 2 3 mean 1 2 3 mean α-santalene 15.10146.4 43.8 45.6 45.3 9.5 9.9 10.1 9.8 α-E-bergamotene 15.288 16.3 15.316.4 16.0 75.7 73.4 74.9 74.7 epi-β-santalene 16.132 4.6 5.1 4.3 4.7 0.90.8 0.5 0.7 β-santalene 16.362 30.4 33.1 31.2 31.5 6.0 7.3 6.4 6.6Z-β-farnesene 16.468 0.7 1.0 0.7 0.8 0.6 0.5 0.5 0.5 E-β-farnesene16.927 1.7 1.7 1.7 1.7 7.4 8.0 7.5 7.6 SauSSy with FPP Mg SauSSy withFPP Mn 1 2 3 mean 1 2 3 mean α-santalene 15.093 51.7 51.5 50.7 51.3 10.310.5 9.7 10.2 α-E-bergamotene 15.278 13.8 14.6 15.2 14.5 72.5 73.8 74.473.6 epi-β-santalene 16.123 4.7 4.6 5.1 4.8 1.4 0.8 1.2 1.2 β-santalene16.352 27.8 27.5 27.0 27.4 5.3 5.5 5.1 5.3 Z-β-farnesene 16.458 0.6 0.60.5 0.6 0.9 0.9 0.9 0.9 E-β-farnesene 16.917 1.5 1.3 1.5 1.4 8.3 8.5 8.68.5 SspiSSy with FPP Mg SspiSSy with FPP Mn 1 2 3 mean 1 2 3 meanα-santalene 15.093 46.3 47.4 48.1 47.3 7.2 7.4 7.3 7.3 α-E-bergamotene15.28 19.6 19.2 18.9 19.2 79.0 78.6 79.1 78.9 epi-β-santalene 16.125 4.64.2 4.0 4.2 1.4 1.7 0.7 1.3 β-santalene 16.354 26.4 26.4 26.3 26.3 3.33.4 3.5 3.4 Z-β-farnesene 16.46 0.7 0.6 0.8 0.7 1.0 1.0 1.0 1.0E-β-farnesene 16.919 2.5 2.2 2.0 2.2 8.1 8.0 8.4 8.2

Modifications of the above-described modes of carrying out the variousembodiments of this invention will be apparent to those skilled in theart based on the above teachings related to the disclosed invention. Theabove embodiments of the invention are merely exemplary and should notbe construed to be in any way limiting.

The invention claimed is:
 1. A method of making a terpene, comprising:a) contacting an acyclic pyrophosphate terpene precursor with a terpenesynthase encoded by a nucleic acid molecule to produce a terpene,wherein: the nucleic acid molecule encodes a Santalum species terpenesynthase that comprises: (i) the sequence of amino acid resides setforth in SEQ ID NO:2, 4 or 6 or a catalytically active fragment thereof;or (ii) a sequence of amino acid residues that has at least 90% sequenceidentity to the sequence of amino acid residues set forth in SEQ ID NO:2 or a catalytically active fragment thereof, wherein the encodedsynthase catalyzes the production of a α-santalene, α-trans-bergamotene,epi-β-santalene and β-santalene concurrently; and if the method isperformed in vivo in a cell, the nucleic acid is heterologous to thecell; and b) processing the terpene that is produced by contacting itwith a P450 enzyme to produce a processed terpene.
 2. The method ofclaim 1, wherein: the terpene synthase is expressed in the cell; and thestep of contacting the acyclic pyrophosphate terpene precursor occurs inthe cell.
 3. The method of claim 2, wherein the acyclic pyrophosphateterpene precursor is expressed in the cell.
 4. The method of claim 2,wherein the terpene is produced in the cell by a method comprisingcultivating the cell under conditions in which the synthase catalyzesthe production of a terpene.
 5. The method of claim 1, wherein theacyclic pyrophosphate terpene precursor is selected from amonggeranyl-diphosphate (GPP), farnesyl-diphosphate (FPP) andgeranylgeranyl-diphosphate (GGPP).
 6. The method of claim 1 that isperformed in vitro.
 7. The method of claim 1, wherein the terpene isselected from among one or more of α-santalene, α-trans-bergamotene,epi-β-santalene and β-santalene.
 8. The method of claim 1, wherein theterpene is selected from among one or more of (+)-epi-β-santalene,(−)-β-santalene, (+)-β-santalene, (+)-α-santalene and (−)-α-santalene.9. The method of claim 7, wherein the terpene is processed to analcohol.
 10. The method of claim 1, wherein the terpene is processed toan alcohol.
 11. The method of claim 10, wherein the alcohol comprisesone or more of α-santalol, β-santalol, α-trans-bergamotol andepi-β-santalol.
 12. The method of claim 1, wherein the processed terpeneis isolated.
 13. The method of claim 1, wherein the terpene is isolatedbefore it is processed.
 14. The method of claim 1, wherein the terpeneproduced in step a) is isolated; and is processed in vitro by contactingit with a P450 enzyme.
 15. The method of claim 1, wherein the synthasecomprises the sequence of amino acids set forth in SEQ ID NO:2.
 16. Themethod of claim 1, wherein the synthase comprises the sequence of aminoacids 32-42, 221-425 or 321-325 of the amino acid sequence set forth inSEQ ID NO:
 2. 17. The method of claim 1, wherein the method is performedin a host cell.
 18. The method of claim 17, wherein the host cell is ayeast cell or a bacterial cell.
 19. The method of claim 1, wherein theterpene synthase comprises the sequence of amino acid resides set forthin SEQ ID NO:2, 4 or 6 or a catalytically active fragment thereof. 20.The method of claim 1, wherein the terpene synthase comprises thesequence of amino acid residues set forth in SEQ ID NO:4.
 21. The methodof claim 1, wherein the terpene synthase comprises the sequence of aminoacid residues set forth in SEQ ID NO:6.